Total RNA Extraction and Purification Total RNA Mini Kit (Tissue)

Total RNA Extraction and Puri cation

Total RNA Mini Kit (Blood/Cultured Cell)...... 47 Total RNA Mini Kit (Tissue)...... 48 Total RNA Mini Kit (Plant)...... 49 Presto TM Mini RNA Bacteria Kit...... 50 Presto TM Mini RNA Yeast Kit...... 51 Presto TM DNA/RNA/Protein Extraction Kit...... 52 miRNA Isolation Kit...... 53 GENEzol TM Reagent...... 56 GENEzol TM TriRNA Bacteria Kit...... 57 GENEzol TM TriRNA Pure Kit...... 58 TriRNA Pure Kit...... 59 RNA Cleanup Kit...... 60 GENEzol TM 96 Well TriRNA Pure Kit...... 61 Total RNA Mini Kit (Blood/Cultured Cell)

Introduction (RB050/100/300, RBD050/100/300) The Total RNA Mini Kit (Blood/Cultured Cell) was designed for total RNA purification from fresh whole human blood and cultured cells. Detergents and chaotropic salt are used to lyse cells and inactivate RNase while RNA is bound by the glass fiber matrix of the RNA spin column. Once any contaminants have been removed, using the Wash Buffer (containing ethanol), the purified total RNA is eluted by RNase-free Water and ready for use in various downstream applications.

Advantages • Purify total RNA within 20 minutes! • High Yield: up to 2 µg from 300 µl whole blood, up to 30 µg from 1x106 293T cells • Sample Volume: up to 300 µl of whole blood, cultured cells (up to 5 x 106) • RNA glass fiber spin columns (membrane optimized for total RNA extraction) • Individually packaged RNA spin columns, certified RNase and DNase-free Total RNA Mini Kit (Blood/Cultured Cell) Protocol Procedure • Elution volume: 25 ~ 100 μl • Storage: dry at room temperature (15-25ºC) for up to 9 months

Applications RT-PCR, Northern Blotting, Primer Extension, mRNA Selection, cDNA Synthesis

Components RBC lysis of whole blood and cultured cell samples • RBC Lysis Buffer • RB Buffer • W1 Buffer • Wash Buffer • DNase I (RBD050, RBD100, RBD300) • DNase I Reaction Buffer (RBD050, RBD100, RBD300) • RNase-free Water Cell lysis of blood and cultured cell samples • RB Columns • 2 ml Collection Tubes

Total RNA Mini Kit (Blood/Cultured Cell) Test Data

Figure 1. Total RNA from 300 µl of whole human blood was RNA binding to membrane while contaminants remain extracted using the Total RNA Mini Kit (Blood/Cultured Cell) and suspended the equivalent competitors kit. The purified RNA was analyzed by electrophoresis on a 1% agarose gel. Lane 1, 2 = Geneaid M = Geneaid 1 Kb DNA Ladder Lane 3, 4 = Competitor

Geneaid Competitor Wash (removal of contaminants while RNA remains bound Yield 260/280 260/230 Yield 260/280 260/230 to membrane) 2.02 µg 1.85 2.15 1.77 µg 1.78 1.42 2.13 µg 1.90 2.35 1.80 µg 1.76 1.19 1 2 M 3 4

Figure 2. Total RNA from 1×106 293T cells was extracted using the Total RNA Mini Kit (Blood/Cultured Cell) and the equivalent Elution of pure total RNA which is ready for subsequent competitors kit. The purified RNA was analyzed by reactions electrophoresis on a 1% agarose gel. Lane 1, 2 = Geneaid M = Geneaid 1 Kb DNA Ladder Lane 3, 4 = Competitor Figure 3. Geneaid RB Columns are specially designed and pretreated to Geneaid Competitor ensure high total RNA yields from blood and cultured cell samples. Yield 260/280 260/230 Yield 260/280 260/230 28.70 µg 1.96 2.32 20.50 µg 1.85 2.31 29.90 µg 1.99 2.02 28.90 µg 1.84 2.32 1 2 M 3 4

Page 47 www.geneaid.com Total RNA Extraction and Purification Total RNA Mini Kit (Tissue)

Introduction (RT050/100/300, RTD050/100/300) The Total RNA Mini Kit (Tissue) was designed for total RNA purification from a variety of animal tissue and paraffin-embedded tissue. Tissue samples can be efficiently homogenized in a microcentrifuge tube using the provided DNase and RNase-free Micropestle. Detergents and chaotropic salt are used to lyse cells and inactivate RNase while RNA is bound by the glass fiber matrix of the RNA spin column. Once any contaminants have been removed, using the Wash Buffer (containing ethanol), the purified total RNA is eluted by RNase-free Water. Phenol extraction or alcohol precipitation is not required and the purified RNA is ready for use in a variety of downstream applications.

Advantages • Purify total RNA within 15 minutes! • High Yield: 5-30 μg of pure RNA • Sample Volume: up to 25 mg of tissue, up to 25 mg of paraffin-embedded tissue • Provided Micropestles are certified DNase and RNase-free Total RNA Mini Kit (Tissue) Protocol Procedure • RNA glass fiber spin columns (membrane optimized for total RNA extraction) • Individually packaged RNA spin columns, certified RNase and DNase-free • Elution volume: 25 ~ 100 μl • Storage: dry at room temperature (15-25ºC) for up to 9 months

Applications RT-PCR, Northern Blotting, Primer Extension, mRNA Selection, cDNA Tissue dissociation in a 1.5 ml microcentrifuge tube using the provided micropestle Synthesis, RNase Protection Assay

Components • Micropestles • RB Buffer • W1 Buffer • Wash Buffer • DNase I (RTD050, RTD100, RTD300) Cell lysis of tissue samples • DNase I Reaction Buffer (RTD050, RTD100, RTD300) • RNase-free Water • RB Columns

Total RNA Mini Kit (Tissue) Test Data

RNA binding to membrane while contaminants remain suspended Figure 1. Total RNA was extracted from 10 mg of various mouse tissue using the Total RNA Mini Kit (Tissue). 5 μl aliquots of each sample were analyzed by electrophoresis on a 1% agarose gel.

Wash (removal of contaminants while RNA remains bound to 1. Brain membrane) 2. Lung 3. Liver 4. Spleen 5. Kidney

1 2 3 4 5 Elution of pure total RNA which is ready for subsequent reactions

Figure 2. Geneaid RB Columns are specially designed and pretreated to ensure high total RNA yields from tissue samples.

Total RNA Extraction and Purification www.geneaid.com Page 48 Total RNA Mini Kit (Plant)

Introduction (RP050/100/300, RPD050/100/300) The Total RNA Mini Kit (Plant) was designed for total RNA purification from plant tissue and cells. RNA from a wide variety of plant species can be purified within 15 minutes. Samples are initially ground in liquid nitrogen then filtered to remove cell debris using the efficient RNA filter columns. In the presence of a binding buffer and chaotropic salt, total RNA in the lysate binds to the glass fiber matrix of the spin column. Once any contaminants have been removed using the Wash Buffer (containing ethanol), the purified total RNA is eluted by RNase-free Water. Phenol extraction or alcohol precipitation is not required and the purified total RNA is ready for use in a variety of subsequent reactions.

Advantages • Purify total RNA within 15 minutes! • High Yield: 5-30 μg of pure RNA • Sample Volume: up to 100 mg (fresh plant tissue), up to 25 mg (dry plant tissue) Total RNA Mini Kit (Plant) Protocol Procedure • RNA glass fiber spin columns (membrane optimized for total RNA extraction) • Individually packaged RNA spin columns, certified RNase and DNase-free • Elution volume: 25 ~ 100 μl • Storage: dry at room temperature (15-25ºC) for up to 9 months

Applications Cell lysis of plant samples RT-PCR, Northern Blotting, Real-time PCR

Components • RB Buffer • PRB Buffer • W1 Buffer • Wash Buffer RNA binding to membrane while contaminants remain • DNase I (RPD050, RPD100, RPD300) suspended • DNase I Reaction Buffer (RPD050, RPD100, RPD300) • RNase-free Water • RB Columns • Filter Columns • 2 ml Collection Tubes Wash (removal of contaminants while RNA remains bound to Total RNA Mini Kit (Plant) Test Data membrane)

Figure 1. Total RNA from 50 mg Thale Cress (Arabidopsis thaliana) leaves was extracted using the Total RNA Mini Kit (Plant). The purified RNA was analyzed by electrophoresis on a 1% agarose gel. Elution of pure total RNA which is ready for subsequent reactions M = Geneaid 1 Kb DNA Ladder

Sample Yield 260/280 260/230 1 27.02 µg 2.07 2.43 2 27.68 µg 2.10 2.40 3 26.15 µg 2.06 2.44 Figure 2. Geneaid RB Columns are specially designed and pretreated to ensure high total RNA yields from a wide variety of plant species. M 1 2 3

Page 49 www.geneaid.com Total RNA Extraction and Purification Presto TM Mini RNA Bacteria Kit

Introduction (RBB050/100/300, RBBD050/100/300) The Presto™ Mini RNA Bacteria Kit was designed for total RNA purification from Gram (-) negative and Gram (+) positive bacteria. The provided Lysozyme and Bacteria Lysis Buffer will efficiently lyse bacterial cell walls consisting of the peptidoglycan layer. Detergents and chaotropic salt are used to further lyse cells and inactivate RNase while RNA is bound by the glass fiber matrix of the RNA spin column. Once any contaminants have been removed, using the Wash Buffer (containing ethanol), the purified total RNA is eluted by RNase-free Water and is ready for use in a variety of subsequent reactions.

Advantages • Purify total RNA within 20 minutes! • High Yield: up to 60 μg of pure RNA (1 x 109 Escherichia coli: 40-45 µg, 1 x 109 Bacillus subtilis: 50-55 µg) • Convenient: Includes Lysozyme and Bacteria Lysis Buffer for easy bacteria lysis Presto™ Mini RNA Bacteria Kit Protocol Procedure • RNA glass fiber spin columns (membrane optimized for total RNA extraction) • Individually packaged RNA spin columns, certified RNase and DNase-free • Elution volume: 50 ~ 100 μl • Storage: dry at room temperature (15-25ºC) for up to 9 months, Lysozyme Cell lysis of bacteria samples is shipped at room temperature and stored at -20ºC for extended periods

Applications RT-PCR, Northern Blotting, Primer Extension, mRNA Selection, cDNA

Synthesis RNA binding to membrane while contaminants remain suspended Components • Bacteria Lysis Buffer • Lysozyme • RB Buffer • W1 Buffer • Wash Buffer Wash (removal of contaminants while RNA remains bound to membrane) • DNase I (RBBD050, RBBD100, RBBD300) • DNase I Reaction Buffer (RBBD050, RBBD100, RBBD300) • RNase-free Water • RB Columns • 2 ml Collection Tubes

Presto™ Mini RNA Bacteria Kit Test Data Elution of pure total RNA which is ready for subsequent reactions

Figure 1. RNA was extracted using the Presto™ Mini RNA Bacteria Kit. An Escherichia coli (1×109) culture (OD600=1.3, 1 ml) was harvested by centrifugation at 16,000 x g for 1 minute. 10 µl aliquots from a 50 µl eluate of purified RNA were analyzed by electrophoresis on a 0.8% agarose gel. Figure 2. Geneaid RB Columns are specially designed and pretreated to ensure high total RNA yields from bacteria samples. Test RNA Yield 260/280 260/230 1 41.56 µg 2.14 2.35 2 40.87 µg 2.15 2.32

1 2

Total RNA Extraction and Purification www.geneaid.com Page 50 Presto TM Mini RNA Yeast Kit

Introduction (RBY050/100/300, RBYD050/100/300) The Presto™ Mini RNA Yeast Kit was designed for total RNA purification from yeast and a wide variety of other fungus species. Sorbitol Buffer is included to reduce sample preparation time and minimize hands on time. Detergents and chaotropic salt are used to lyse cells and inactivate RNase while RNA is bound by the glass fiber matrix of the RNA spin column. Once any contaminants have been removed, using the Wash Buffer (containing ethanol), the purified total RNA is eluted by RNase-free Water. The entire procedure can be completed within 30 minutes and the purified RNA is ready for use in RT-PCR, Northern Blotting, Primer Extension, mRNA Selection and cDNA Synthesis.

Advantages • Purify total RNA within 20 minutes! • High Yield: 5-30 μg of pure RNA • Convenient: Includes premixed Sorbitol Buffer to speed up sample preparation when purifying RNA from yeast other fungus species Presto™ Mini RNA Yeast Kit Protocol Procedure • RNA glass fiber spin columns (membrane optimized for total RNA extraction) • Individually packaged RNA spin columns, certified RNase and DNase-free • Elution volume: 50 ~ 100 μl • Storage: dry at room temperature (15-25ºC) for up to 9 months Cell lysis of yeast and other fungi samples

Applications RT-PCR, Northern Blotting, Primer Extension, mRNA Selection, cDNA Synthesis

RNA binding to membrane while contaminants remain Components suspended • Sorbitol Buffer • RB Buffer • W1 Buffer • Wash Buffer • DNase I (RBYD050, RBYD100, RBYD300) • DNase I Reaction Buffer (RBYD050, RBYD100, RBYD300) Wash (removal of contaminants while RNA remains bound to • RNase-free Water membrane) • RB Columns • 2 ml Collection Tubes

Presto™ Mini RNA Yeast Kit Test Data

Elution of pure total RNA which is ready for subsequent Total RNA was extracted using the Presto™ Mini Figure 1. reactions RNA Yeast Kit. Saccharomyces cerevisiae (5×107) was harvested by centrifugation at 5,000 x g for 10 minutes. 5 µl aliquots of purified total RNA from a 50 µl eluate were analyzed by electrophoresis on a 0.8% agarose gel.

M = Geneaid 1 Kb DNA Ladder Figure 2. Geneaid RB Columns are specially designed and pretreated to Test RNA Conc. 260/280 260/230 Yield ensure high total RNA yields from yeast and other fungus species. 1 391.0 µg/ml 2.19 2.48 19.6 µg 2 389.9 µg/ml 2.19 2.51 19.5 µg 3 387.9 µg/ml 2.20 2.51 19.4 µg

M 1 2 3

Page 51 www.geneaid.com Total RNA Extraction and Purification Presto TM DNA/RNA/Protein Extraction Kit

Introduction (DRP050, DRP100) The PrestoTM DNA/RNA/Protein Extraction Kit provides an efficient method for purifying genomic DNA, total RNA and total protein simultaneously from a variety of samples (cultured cells, animal tissue, whole blood and biological liquids). Chaotropic salt is used to lyse cells and inactive DNases and RNases, allowing DNA to bind to the genomic DNA spin column. The flow-through can then be transferred to the RNA spin column for RNA binding. The proteins in the flow-through can then be precipitated using acetone. Contaminants are effectively removed using wash buffers followed by pure genomic DNA elution in a low salt buffer and pure total RNA elution in RNase-free Water. DNA/RNA purification can be completed in 25 minutes without phenol/chloroform extraction or alcohol precipitation and protein purification can be completed in 50 minutes. The purified DNA, with approximately 20-30 Kb, is suitable for use in PCR or other enzymatic reactions and the purified RNA (including miRNA) is ready for use in RT-PCR, Real-time PCR, northern blotting, primer extension, mRNA selection and cDNA synthesis. The purified proteins can be directly analyzed on a SDS-PAGE and subsequent western blot.

Advantages • Purify genomic DNA and total RNA in 25 minutes! Presto™ DNA/RNA/Protein Extraction Kit Protocol Procedure • High yield: up to 9 μg of genomic DNA, 20 μg of total RNA, 120 μg of protein from 1.5 x 106 HeLa cells • Sample: cultured cells, animal tissue, whole blood, biological fluids Sample preparation and • Format: genomic DNA spin column and total RNA spin column certified Cell lysis DNase and RNase-free • Elution volume: 50-200 μl (genomic DNA) / 25-50 μl (total RNA) • Storage: dry at room temperature (15-25ºC) for up to 9 months • Cost effective DNA/RNA Separation: DNA binding and RNA Applications flow-through The purified DNA, approximately 20-30 kb, is suitable for use in PCR or other enzymatic reactions and the purified RNA (including miRNA) is ready for use in RT-PCR, Real-time PCR, northern blotting, primer extension, mRNA selection and cDNA synthesis. Protein Protein RNA Components DNA flow-through Precipitation binding • RBC Lysis Buffer wash on ice for 30 • DR Buffer minutes • RW1 Buffer • RPE Buffer • W1 Buffer • Wash Buffer • RNase-free Water DNA RNA º • Elution Buffer (10 mM Tris-HCl, pH8.5 at 25 C) elution Protein wash • GD Columns Resuspension • RB Columns • 2 ml Collection Tubes

Presto™ DNA/RNA/Protein Kit Functional Test Data

6 Figure 1. Genomic DNA and Total RNA from 1 x 10 SSN-1 cells RNA was extracted using the Presto™ DNA/RNA/Protein Extraction 100ºC for 3-5 elution Kit. 10 µl from 100 µl eluates of purified genomic DNA and 10 µl minutes from 50 µl eluates of purified total RNA was analyzed by electrophoresis on a 1% agarose gel. 1-2 = DNA from 1 x 106 SSN-1 cells 3-4 = RNA from 1 x 106 SSN-1 cells M = Geneaid 1 Kb DNA Ladder SDS-PAGE Analysis

Sample µg/ml 260/280 260/230 Yield 1. DNA 48.3 1.92 2.10 4.83 µg 2. DNA 46.7 1.92 1.99 4.67 µg 3. RNA 432.8 2.11 2.24 21.64 µg Figure 2. Geneaid GD Columns are specially designed and pretreated to 4. RNA 426.7 2.11 2.22 21.34 µg ensure high genomic DNA yields from a variety of samples. Geneaid RB Columns are specially designed and pretreated to ensure high total RNA yields from a variety of samples. M 1 2 3 4

Total RNA Extraction and Purification www.geneaid.com Page 52 miRNA Isolation Kit

Introduction (RMI050) The miRNA Isolation Kit provides a quick and easy spin column system for purifying and enriching micro RNAs (miRNAs) and other small cellular RNAs from a wide variety of tissue and cells. Since miRNAs are vital for regulating gene expression, this kit is optimized for isolation of small RNA molecules while removing larger RNAs and minimizing genomic DNA contamination for improved sensitive downstream applications.

Advantages • Purify miRNA within 30 minutes! • Efficient removal of larger RNA for sensitive downstream applications • Sample: 100 mg of tissue, cultured cells (up to 1 x 106) • Phenol/chloroform/spin column miRNA purification • Provided Micropestles are certified DNase and RNase-free • RNA glass fiber spin columns optimized for miRNA extraction • Individually packaged RNA spin columns, certified RNase and DNase-free • Elution volume: 50-100 μl miRNA Isolation Kit Protocol Procedure • Storage: dry at room temperature (15-25ºC) for up to 9 months

Applications miRNA analysis, siRNA analysis, shRNA analysis and snRNA analysis, Cell lysis qPCR, microarray analysis

Components • Lysis Buffer • Mi Buffer Larger RNA binding to membrane while contaminants and • Wash Buffer miRNA easily flow-through • Release Buffer • Micropestles • RNA Columns • 2 ml Collection Tubes miRNA Isolation Kit Comparison Data Ethanol treatment of miRNA to facilitate binding to miRNA Kit Liver Brain Muscle Spleen membrane Threshold Geneaid 23 22.4 21.3 21.8 Cycle (Ct) Brand Q 23.5 23.2 21.7 22.1 Table 1. Comparison of mouse miRNA purified using Geneaid and Brand Q kits. miRNA was polyadenylated with E. coli Poly A RNA polymerase in the presence of ATP. The miRNA-poly(A) was then used as a template for the synthesis of Universal cDNA and then the cDNA was used miRNA binding to membrane while contaminants remain as templates for the Internal Control (5S RNA). The primers used were h5SA/41R target in the suspended N-terminal of 5S RNA. The threshold cycles (Ct) were determined and used for the performance comparison.

Wash (removal of ethanol and contaminants while miRNA For miRNA Isolation Kit test data, please refer to remain bound to membrane) page 46 and 47.

Elution of pure miRNA which is ready for subsequent reactions

Figure 3. Geneaid RNA Columns are specially designed and pretreated to ensure high miRNA yields from a wide variety of samples of tissue and cell samples.

Page 53 www.geneaid.com Total RNA Extraction and Purification miRNA Isolation Kit

miRNA Isolation Kit Test Data

Brain Muscle Spleen Brain Muscle Spleen

28S Figure 1. Total RNA from mouse tissue was isolated using a reagent system. 18S 10 µg of each RNA was subjected to miRNA isolation using the miRNA Isolation Kit. The purified miRNA was resolved in 50 µl of Release Buffer and a 1/10 volume aliquot (5 µl) was analyzed by electrophoresis on a 2% agarose gel.

5S/tRNA

Total RNA Purified miRNA

700 500 Figure 2. Small RNAs were isolated from 4 human placental samples. Each lane contains 1/10 of the purified miRNA. 300

100 tRNA miRNA

Threshold Cycle Amplification Plot Standard Curve

Rn miRNA E7 E6 E5 E4 E3 Sample

Threshold Cycle

Log Starting Quantity, Copy Number E = 129.7%, R^2 = 0.996, slope = -2.768, y-int = 27… FAM

Figures 3 and 4. Quantitation of miR16 RNA by SYBR qPCR. miR16 RNA quantitation PCR used miRNA16ssIC/dT25 primers to target specifically the miR16 RNA. The Universal cDNA reverse transcribed by dT25 on miRNA-poly(A) template was used as the qPCR target.

Total RNA Extraction and Purification www.geneaid.com Page 54 miRNA Isolation Kit

Threshold Cycle Amplification Plot Standard Curve Rn miRNA E7 E6 E5 E4 E3 Sample

E7 E6 E5 E4 E3 N Threshold Cycle

Cut-off

Log Starting Quantity, Copy Number E = 99.3%, R^2 = 0.997, slope = -3.338, y-int = 31… FAM

Figures 5 and 6. Quantitation of miR25 RNA by SYBR qPCR. miR25 RNA quantitation PCR used miRNA25ssIC/dT25 primers to specifically target the miR25 RNA. The Universal cDNA reverse transcribed by dT25 on miRNA-poly(A) template was used as the qPCR target.

5S RNA

Figure 7. Quantitation of miRNA expressed in various mouse tissues. miRNA was purified using the Geneaid miRNA Isolation kit. The miR16 RNA miRNA-poly(A) was then used as a template for the synthesis of Universal cDNA. The cDNA was then used as a template for the Internal Control (5S RNA) and individual miRNA qPCR assays. The reverse primer used in the qPCR was dT25.

1 = Mouse Liver 2 = Mouse Brain 3 = Mouse Muscle 4 = Mouse Spleen

miR25 RNA

Page 55 www.geneaid.com Total RNA Extraction and Purification GENEzol TM Reagent

Introduction (GZR050, GZR100, GZR200) GENEzol™ Reagent is a phenol, chloroform and guanidine isothiocyanate based scalable solution for extracting high-quality total RNA as well as simultaneous extraction of RNA, DNA and protein from a wide variety of samples such as blood, buffy coat, plasma, serum, cultured cells and tissue. The extracted RNA can be used directly in a variety of downstream applications such as cDNA Library Construction, Cloning, RT-PCR (Endpoint), Real-Time PCR, Nuclease Protection Assays and Northern Blotting. GENEzol™ Reagent is a cost effective alternative for efficient total RNA extraction.

Advantages • Extract total RNA or simultaneous RNA, DNA and protein within 1 hour • Sample: up to 300 µl (blood, buffy coat, serum, plasma), up to 5 x 106 (cultured cells), 50-100 mg (tissue) • Scalable • Format: phenol, chlorophorm and guanidine isothiocyanate GENEzol™ Reagent Real-Time PCR Data

• Storage: 2ºC to 25ºC for up to 12 months Amplification Chart 1200 • Cost effective Trizol® alternative RNA whole blood (5.2x1010 copy/ml) 1000 RNA standard Applications (107 copy/ml) 800 cDNA Library Construction, Cloning, RT-PCR (Endpoint), Real-Time PCR, RNA standard (106 copy/ml) Nuclease Protection Assays and Northern Blotting 600 RNA standard 5 400 (10 copy/ml) Components 200 RNA standard • GENEzol™ Reagent (104 copy/ml) 23.88 -0 PCR Base Line Subtracted Fit RFU 0 10 20 30 GENEzol™ Reagent Test Data Cycle

Figure 2. Quantitative analysis of human beta globin mRNA extracted by GENEzol™ TriRNA Pure Kit using a Taqman probe 1-step qRT-PCR assay. Figure 1. RNA from a 1 ml human blood sample was extracted The assay was run on a BioRad IQ5 thermal cycler. The high yield, high using GENEzol™ Reagent. 10 µl from a 50 µl eluate of RNA was quality extracted RNA was amplified quickly following a very short C analyzed by electrophoresis on a 0.8% agarose gel. T M = Geneaid 1 Kb DNA Ladder (threshold cycle) compared to the RNA standards.

Test RNA Conc. 260/280 Yield 1 119.9 µg/ml 1.82 6.0 µg 2 135.5 µg/ml 1.76 6.8 µg 3 176.1 µg/ml 1.77 8.8 µg

M 1 2 3

GENEzolTM Reagent DNA/RNA/Protein Extraction

Total RNA Extraction and Purification www.geneaid.com Page 56 GENEzol TM TriRNA Bacteria Kit

Introduction (GZB050, GZB100) GENEzol™ TriRNA Bacteria Kit is a phenol, chloroform and guanidine isothiocyanate based scalable solution for extracting high-quality total RNA as well as simultaneous extraction of RNA, DNA and protein from bacteria samples. The provided Lysozyme and Bacteria Lysis Buffer will efficiently lyse bacterial cell walls consisting of the peptidoglycan layer. The extracted RNA can be used directly in a variety of downstream applications such as cDNA Library Construction, Cloning, RT-PCR (Endpoint), Real-Time PCR, Nuclease Protection Assays and Northern Blotting. GENEzol™ TriRNA Bacteria Kit is a cost effective alternative for efficient total RNA extraction.

Advantages • Purify total RNA or simultaneous RNA, DNA and protein within 1 hour • Sample: bacteria cells up to 1 x 109 • Scalable • Format: phenol, chloroform and guanidine isothiocyanate • Convenient: includes Lysozyme and Bacteria Lysis Buffer GENEzol™ TriRNA Bacteria Kit Protocol Procedure

Applications cDNA Library Construction, Cloning, RT-PCR (Endpoint), Real-Time PCR, Nuclease Protection Assays and Northern Blotting Sample preparation and bacteria cell lyis Components and Storage • GENEzol™ Reagent is shipped at room temperature and stored dry at 2ºC to 25ºC for up to 12 months • Bacteria Lysis Buffer is shipped at room temperature and stored dry at room temperature (15-25ºC) for up to 12 months • Lysozyme is shipped at room temperature and stored at -20ºC for up to 12 months Phase Separation GENEzol™ TriRNA Bacteria Kit Test Data

Figure 1. RNA was extracted using the GENEzol™ TriRNA Bacteria Kit. An Escherichia coli (1×109) culture (OD600=1.3, 1 ml) was harvested by centrifugation at 16,000 x g for 2 minutes, followed by RNA extraction. 10 µl aliquots from a 50 µl eluate of RNA precipitation RNA were analyzed by electrophoresis on a 0.8% agarose gel.

Test RNA Conc. 260/280 Yield 1 589.0 µg/ml 1.90 29.5 µg 2 616.9 µg/ml 1.88 30.8 µg 3 578.4 µg/ml 1.84 28.9 µg

RNA Wash

RNA Re-suspension

Figure 2. GENEzol™ Reagent uses a simple procedure to extract high-quality total RNA from bacteria samples.

Page 57 www.geneaid.com Total RNA Extraction and Purification GENEZol TM TriRNA Pure Kit

Introduction (GZX050/100/200, GZXD050/100/200) The GENEzol™ TriRNA Pure Kit is a phenol and guanidine isothiocyanate plus spin column system for convenient purification of high-quality total RNA from a variety of samples. Initially samples are homogenized in GENEzol™ Reagent without chloroform phase separation or isopropanol RNA precipitation. Following sample homogenization, simply bind, wash and elute.

Advantages • Purify total RNA within 15 minutes! • Sample: up to 200 µl (blood, buffy coat, serum, plasma), up to 5 x 106 (cultured cells), 50-100 mg (tissue), up to 1 x 109 (bacteria cells), 20-50 mg (plant tissue) • Homogenize samples with GENEzol™ Reagent then bind, wash and elute • No phase separation, No precipitation, No phenol carryover • High Yield: 50 µg RNA from ≥ 25 µl DNase/RNase-free Water • Format: phenol, guanidine isothiocyanate solution and spin column • Storage: GENEzol™ Reagent is stored dry at 2ºC to 25ºC for up to 9 GENEzol™ TriRNA Pure Kit Real-Time PCR Data Amplification Chart months, the other componets are stored dry at room temperature (15-25ºC) 1200 RNA whole blood for up to 9 months (5.2x1010 copy/ml) 1000 RNA standard (107 copy/ml) Applications 800 RNA standard (106 copy/ml) cDNA Library Construction, Cloning, RT-PCR (Endpoint), Real-Time PCR, 600 RNA standard Nuclease Protection Assays and Northern Blotting 5 400 (10 copy/ml)

Components 200 RNA standard (104 copy/ml) • GENEzol™ Reagent, W1 Buffer, Wash Buffer, RNase-free Water, RB Columns, 2 ml 23.88 -0 Collection Tubes, DNase I and DNase I Reaction Buffer (GZXD050/100/300) PCR Base Line Subtracted Fit RFU 0 10 20 30 Cycle GENEzol™ TriRNA Pure Kit Test Data Figure 2. Quantitative analysis of human beta globin mRNA extracted by GENEzol™ TriRNA Pure Kit using a Taqman probe 1-step qRT-PCR assay. Figure 1. RNA was purified using the GENEzol™ TriRNA Pure The assay was run on a BioRad IQ5 thermal cycler. The high yield, high Kit. An Escherichia coli (1×109) culture (OD600=1.3, 1 ml) was quality extracted RNA was amplified quickly following a very short CT harvested by centrifugation at 16,000 x g for 2 minutes, followed (threshold cycle) compared to the RNA standards. GENEzol™ Reagent homogenization. RNA was then purified using a spin column procedure. 10 µl aliquots from a 50 µl eluate of purified RNA were analyzed by electrophoresis on a 0.8% agarose gel. No chlorophorm phase separation

Test RNA Concentration 260/280 260/230 Yield No isopropanol RNA precipitation 1 350.3 µg/ml 2.03 2.24 17.5 µg 2 422.3 µg/ml 2.08 2.33 21.1 µg No phenol carryover 3 372.1 µg/ml 2.04 2.24 18.6 µg

1 2 3

GENEzolTM Reagent TriRNA Pure Kit

Total RNA Extraction and Purification www.geneaid.com Page 58 TriRNA Pure Kit

Introduction (TRP050/100/200, TRPD050/100/200) The TriRNA Pure Kit is a spin column system for purification of high-quality total RNA from a variety of samples. Initially, samples are homogenized in GENEzol™ Reagent or other phenol, guanidine isothiocyanate reagent. Chloroform phase separation or isopropanol RNA precipitation is not required. Following sample homogenization, simply bind, wash and elute.

Advantages • Purify total RNA within 10 minutes! • Homogenize samples with GENEzol™, TRI-Reagent®, TRIzol®, RNAzol®, QIAzol® etc. • Sample: up to 200 µl (blood, buffy coat, serum, plasma), up to 5 x 106 (cultured cells), 50-100 mg (tissue), up to 1 x 109 (bacteria cells), 20-50 mg (plant tissue) • Simply bind, wash and elute. • No phase separation, No precipitation, No phenol carryover • High Yield: 50 µg RNA from ≥ 25 µl DNase/RNase-free Water TriRNA Pure Kit Protocol Procedure • Format: phenol, guanidine isothiocyanate solution and spin column • Storage: dry at room temperature (15-25ºC) for up to 9 months

Applications Sample preparation using GENEzol™, TRI-Reagent®, cDNA Library Construction, Cloning, RT-PCR (Endpoint), Real-Time PCR, TRIzol®, RNAzol®, QIAzol® etc. without chloroform phase separation or isopropanol RNA precipitation Nuclease Protection Assays and Northern Blotting

Components • W1 Buffer • Wash Buffer • RNase-free Water RNA binding to membrane while contaminants remain suspended • RB Columns • 2 ml Collection Tubes

TriRNA Pure Kit Functional Test Data

Figure 1. RNA was purified using the TriRNA Pure Kit. An Escherichia coli (1×109) culture (OD600=1.3, 1 ml) was Wash (removal of contaminants while RNA remains bound to harvested by centrifugation at 16,000 x g for 2 minutes, followed membrane) GENEzol™ Reagent homogenization. RNA was then purified using a spin column procedure. 10 µl aliquots from a 50 µl eluate of purified total RNA were analyzed by electrophoresis on a 0.8% agarose gel.

Test RNA Concentration 260/280 260/230 Yield 1 350.3 µg/ml 2.03 2.24 17.5 µg Elution of pure total RNA which is ready for subsequent 2 422.3 µg/ml 2.08 2.33 21.1 µg reactions 3 372.1 µg/ml 2.04 2.24 18.6 µg Table 1. RNA purified using the TriRNA Pure Kit.

1 2 3 Figure 2. Geneaid RB Columns are specially designed and pretreated to ensure high total RNA yields from contaminated RNA samples. Test RNA Concentration 260/280 260/230 Yield 1 58.6 ng/µl 1.86 1.81 2.1 µg 2 68.1 ng/µl 1.87 1.81 2.4 µg 3 74.2 ng/µl 7.87 1.83 2.6 µg

Table 2. RNA was purified using the TriRNA Pure Kit. RNA from 200 µl of whole human blood (3 donors) was initially homogenized using GENEzol™ Reagent. RNA was then purified using a spin column procedure. 10 µl from each 35 µl eluate of purified RNA was analyzed by electrophoresis on a 0.8% agarose gel. M = Geneaid 1 Kb DNA Ladder

Page 59 www.geneaid.com Total RNA Extraction and Purification RNA Cleanup Kit

Introduction (PR050, PR100) The RNA Cleanup Kit uses a simple and efficient spin column procedure to purify Total RNA stored in RNase-free water, elution buffer or TE Buffer following extraction using acid-guanidinium-phenol-chlorofom based methods such as TRIzol® Reagent and Geneaid’s GENEzol™ Reagent. Contaminants such as RNases, DNA and residual phenol are effectively removed using a simple 4 step procedure. The high-quality, total RNA is eluted in RNase-free Water or TE (RNase-free) and is ready for use in a variety of sensitive downstream applications.

Advantages • Purify up to 50 µg of total RNA within 10 minutes • Recovery: up to 80% of high quality RNA (A260/A280 = 1.9-2.0) • Elution volume: 20-50 μl • Compatibility: purify RNA stored in RNase-free water, elution buffer or TE Buffer following extraction using GENEzol™, TRI-Reagent®, TRIzol®, RNAzol® and QIAzol® etc. RNA Cleanup Kit Protocol Procedure • Storage: dry at room temperature (15-25ºC) for up to 9 months

Applications cDNA Library Construction, Cloning, RT-PCR (Endpoint), Real-Time PCR, Sample stored in elution buffer following extraction using Nuclease Protection Assays and Northern Blotting GENEzol™, TRI-Reagent®, TRIzol®, RNAzol®, QIAzol® etc. Components • RNA Pure Buffer Buffer • Wash Buffer • RNase-free Water • RB Columns RNA binding to membrane while contaminants remain • 2 ml Collection Tubes suspended

RNA Cleanup Kit Test Data Figure 1. Total RNA was extracted using Supplier I Tri Reagent. An Genomic DNA Escherichia coli (1×109) culture (OD600=1.3, 1 ml) was harvested by centrifugation at 16,000 x g for 1 minute. 10 µl from a 50 µl eluate of Wash (removal of contaminants while RNA remains bound to extracted RNA was analyzed by electrophoresis on a 0.8% agarose gel. membrane) Genomic DNA and other contaminants remain in the final product. M = Geneaid 1 Kb DNA Ladder Test 260/280 260/230 Yield 1 1.67 0.72 21.3 µg 2 1.60 0.84 20.2 µg 3 1.63 1.07 22.4 µg Figure 2. Total RNA was extracted using Supplier I Tri Reagent then Elution of pure total RNA which is ready for subsequent No Genomic DNA purified using the RNA Pure Kit. 10 µl from a 50 µl eluate of purified RNA reactions was analyzed by electrophoresis on a 0.8% agarose gel. Genomic DNA and other contaminants are removed. M = Geneaid 1 Kb DNA Ladder Test RNA Conc. 260/280 260/230 Yield Recovery 1 335.7 µg/ml 1.97 2.44 16.8 µg 78.9% 2 287.2 µg/ml 1.93 2.42 14.4 µg 71.3% Figure 3. Geneaid RB Columns are specially designed and pretreated to 3 330.9 µg/ml 1.96 2.43 16.5 µg 73.7% ensure high total RNA yields from contaminated RNA samples.

Total RNA Extraction and Purification www.geneaid.com Page 60 GENEzol TM 96 Well TriRNA Pure Kit

Introduction (96GZX04, 96GZX10) The GENEzol™ 96 Well TriRNA Pure Kit is a phenol and guanidine isothiocyanate plus 96 well RNA binding plate system for high-throughput purification of high-quality total RNA from a variety of samples. Initially, samples are homogenized in GENEzol™ Reagent without chloroform phase separation or isopropanol RNA precipitation. Following sample homogenization, simply bind, wash and elute the high-quality, total RNA in RNase-free Water and use in a variety of sensitive downstream applications.

Advantages • No chlorophorm phase separation • No isopropanol RNA precipitation • No phenol carryover • Samples: blood, buffy coat, serum, plasma, cultured cells, tissue, bacterial cells, plant tissue • High quality RNA: A260/A280 >1.8, A260/A230 >1.8 • Binding Capacity: 50 µg RNA per well • Binding Plates: glass fiber membrane optimized for total RNA extraction 96 Well GENEzol™ TriRNA Pure Kit Protocol Procedure (certified RNase and DNase-free) • Storage: GENEzol™ Reagent should be stored at 2ºC to 25ºC for up to 9 months, other components should be stored at room temperature (15-25ºC) Transfer for up to 9 months GENEzol™ Reagent treated Applications samples to 96 cDNA Library Construction, Cloning, RT-PCR (Endpoint), Real-Time PCR, Well RNA Nuclease Protection Assays and Northern Blotting Binding plate

Components

• GENEzol™ Reagent • Pre-Wash and Wash Buffer • RNase-free Water • Presto™ 96 Well RNA Binding Plates • Microtubes and Caps RNA binding • 96 Deep Well and 0.35 ml Plates

• Adhesive Film

GENEzol™ 96 Well TriRNA Pure Kit Test Data (HeLa Cells)

Figure 1. 5 x 105 HeLa cells were homogenized using GENEzol™ Reagent and competitor Z tri reagent. RNA was then purified using the corresponding kits binding plate procedure. 10 µl from a 50 µl eluate of purified RNA was analyzed by electrophoresis on a 0.8% agarose gel. Product Test ng/µl 260/280 260/230 Yield Wash 1. Competitor Z 162.5 2.00 2.07 8.1 µg 2. Competitor Z 160.7 2.03 2.07 8.0 µg 3. Geneaid 164.0 2.00 2.07 8.2 µg 4. Geneaid 161.6 2.03 2.06 8.0 µg

GENEzol™ 96 Well TriRNA Pure Kit Real-Time PCR Data Amplification Chart 1200 RNA was extracted from 200 µl of whole human blood (5.2 x 1010 copy/ml) using GENEzol™ 96 Well TriRNA 1000 Pure Kit.

800 RNA standard (107 copy/ml)

RNA standard (106 copy/ml)

600 RNA standard (105 copy/ml)

400 Elution of pure RNA into 0.35 ml 200 RNA standard (104 copy/ml) Collection Plate 23.88 PCR Base Line Subtracted-0 Fit RFU

0 10 20 30 Cycle Figure 2. Quantitative analysis of human beta globin mRNA extracted by GENEzol™ 96 Well TriRNA Pure Kit using a Taqman probe 1-step qRT-PCR assay. The assay was run on a BioRad IQ5 thermal cycler. The high yield, high quality extracted RNA was amplified quickly following a Figure 3. GENEzolTM 96 Well TriRNA Pure Kits can be used efficiently with very short CT (threshold cycle) compared to the RNA standards. either vacuum or centrifuge.

Total RNA Extraction and Purification www.geneaid.com Page 61