RNA Amplification Protocol

RNA amplification protocol

This method is to generate from minute amount of total RNA, sufficient amount of mRNA to perform microarray experiments

You will need: Total RNA If possible use 3 µg, For a better success, clean the Trizol extracted total RNA using the RNeasy kit. To Save columns: If your plan is to pool your RNA samples. Pool first than purify

- Using less than 0.5 µg of starting RNA is more challenging, you will need to perform a second round of amplification. - 1 µg is enough - More than 2 µg is theoretically better

Oligo-dT-T7 5' - GCA TTA GCG GCC GCG AAA TTA - Must be PAGE Purification primer ATA CGA CTC ACT ATA GGG AGA TTT - Reference: Baugh LR, Hill A. A., TTT TTT TTT TTT TTT TTT V - 3' Brown E. L. and Hunter CP (2001) Quantitative analysis of mRNA Make a 500 ng/µl solution, use RNase free amplification by in vitro transcription . water Nucleic Acids Research, Vol. 29, No. 5 e29. Random 5’ NNNNNN 3’ hexamers Make a 1µg/µl solution, use RNase free water Mmlv Promega 200 units/µl Good enough, cheaper than the superscript II Superscript II or Invitrogen 200 units/µl Most used/cited in articles III Is supposed to generate full length cDNAs dNTPs ISC Bioexpress, make 10 mM stocks rNTPs NEB (80 mM solutions, use as it is) E.coli DNA NEB, 10 units/µl, works with NEB buffer #2 polymerase E.coli Rnase H Takara (2150A), 60 units/µl E.coli DNA NEB, 10 units/µl ligase T4 DNA NEB, 3 units/µl polymerase T7 RNA Fermentase, 200 units/µl polymerase Pyrophosphatas Fermentas, 0.1 units/µl e HEPES 1M pH 7.5 Spermidine Make 100 mM stocks, store at -20C 100mM Dithiothreitol Make 1M stocks, store at -20C (DTT) 1M Acetyled BSA 10mg/ml (100X) RNeasy kit Qiagen PCR qiaquick Qiagen purification kit RNase free Use the RNase free water that comes with the Water RNeasy kit Filter tips Speed vacuum Is in the Genelab PCR machine Incubate your reaction in 0.2 ml tubes Set the heated lid at 37C when performing the overnight invitro transcription

There are three steps: 1. Step1: First strand cDNAs synthesis by reverse transcription (the products are single stranded cDNAs). The T7 promoter is added to the cDNAs synthesized by the use of the oligo-dT-T7 primer.

2. Step2: Second strand synthesis (the products are double stranded DNA molecules) The T7 promoter need to be double stranded to be functional.

3. Step 3: The Invitro transcription: the T7 RNA polymerase will transcribe the cDNAs into mRNAs .

If necessary (you do not obtain a sufficient yield for all the microarray hybridizations you plan), perform a second round of amplification.

1. Step1: First strand cDNAs synthesis

Follow the next procedures, use 0.2 ml PCR tubes, filter tips, do the incubation in the PCR machine .

Total RNA 3 µg Oligo-dT-T7 primer (500ng/µl) 1 µl H2O Up to 15 µl

Incubate for 5 min at 70 C, then immediately chill on ice (leave on ice for 10 min). Spin the tubes, and add 10 µl of the following mixture (prepare a master mix for the number of samples you want to amplify): Final volume is 25 µl.

5 X First strand Buffer (contain 5 µl DTT)* dNTPs 10 mM 1.25 µl MMLV (200 units/µl) 1 µl RNase inhibitor 0.5 µl H2O 2.25 µl * some buffer do not contain DTT, in this case add DTT to 10 mM final concentration

Mix gently by pipetting up and down, and incubate at 42C for 1hour to 1hour 30min.

Inactivate the reverse transcriptase by incubating the reaction at 70C for 10 min.

Spin the tubes.

2. Step2: Second strand synthesis (the products are double stranded DNA molecules)

Add 75 µl of the following mixture to the RT reactions: Final volume is 100 µl E.coli DNA polymerase buffer (10X) (use the 10 µl NEB buffer #2) dNTPs 10 mM 2.5 µl E.coli Rnase H (60 units/µl) 0.05 µl (3 units) E.coli DNA polymerase (10 units/µl) 4 µl (40 units) H2O Up to 75 µl Incubate at 16C for 2hours

Then, add: E.coli DNA ligase 0.1µl (1 unit) DNA ligase buffer 11 µl 10x

Incubate at RT for 15 min

Then add: T4 DNA polymerase 2 µl (6 units) Incubate for 15 min at RT

Purify the double strand cDNAs using the qiagen qiaquick PCR purification kit.

To improve the elution yield, use a bigger volume of H2O (two times 50 µl) than mentioned in the qiagen manual. Speed-vacuum to dry.

3. Step 3: Invitro transcription

Add 40 µl of the following invitro transcription cocktail to the double strand cDNAs that you speed vacuumed: HEPES 1 M pH 7.5 4.8 µl (120 mM) MgCl2 2M 0.56 µl (28 mM) rNTPs 80 mM 3 µl (6 mM) Spermidine 100 mM 0.4 µl (1 mM) Acetyled BSA 10X 0.4 µl (0.1 mM) DTT 1M 1.6 µl (40 mM) RNase inhibitor 0.5 µl Pyrophosphatase 2 µl (0.2 units) T7 RNA polymerase 1 µl (200 units) H2O 25.74 µl

Incubate for 14 hours at 37 C (SET THE PCR MACHINE HEATED LID TEMPERATURE AT 37C)

Purify the amplified RNA using the qiagen RNeasy kit. Elute two times with 50 µl RNase free water each time.

Speed vacuum to concentrate (for example until 30 µl are left)

Quantify using the nanodrop

If you did not generate a sufficient amount of amplified RNA, perform a second round of amplification.

Second round of amplification

Carry out the 3 above steps, but with the following modifications:

Step1: Perform the RT reactions using the random hexamers primers (5’NNNNNN3’) instead of the oligo-dT-T7 primer Total RNA 3 µg Random primers 1µg/µl 1 µl H2O Up to 15 µl

Incubate at 70C for 5 min then immediately chill on ice and leave for 10 min at room temperature. Spin the tubes, and add 10 µl of the following mixture (prepare a master mix for the number of samples you want to amplify):

Mix gently by pipetting up and down, and incubate at 37 C for 1 hour.

Then, incubate for 2 min at 94 C, chill rapidly on ice.

Spin the tubes.

2. Step2: Second strand synthesis (the products are double stranded DNA molecules)

Add: Oligo-dT-T7 primer (500 ng/µl) 1 µl

Incubate at 70 C for 5 min, then 3 min at 42 C

Add 74 µl of the following mixture (final volume = 100 µl) E.coli DNA polymerase buffer (10X) (use the 10 µl NEB buffer #2) dNTPs 10 mM 2.5 µl E.coli Rnase H 0.05 µl (3 units) E.coli DNA polymerase 4 µl (40 units) H2O Up to 74 µl Incubate at 16C for 2hours

Then, add: T4 DNA polymerase 2 µl (6 units) Incubate at RT for 15 min

Purify the double strand cDNAs using the qiagen qiaquick PCR purification kit.

Speed vacuum until dry

3. Step 3: Invitro transcription

Add 40 µl of the following invitro transcription cocktail to the double strand cDNAs that you speed vacuumed: HEPES 1 M pH 7.5 4.8 µl (120 mM) MgCl2 2M 0.56 µl (28 mM) rNTPs 80 mM 3 µl Spermidine 100 mM 0.4 µl (1 mM) Acetyled BSA 10X 0.4 µl (0.1 mM) DTT 1M 1.6 µl (40 mM) RNase inhibitor 0.5 µl Pyrophosphatase 2 µl (0.2 units) T7 RNA polymerase 1 µl (200 units) H2O 25.74 µl

Incubate for 14 hours at 37 C (SET THE PCR MACHINE HEATED LID TEMPERATURE AT 37C)

Purify the amplified RNA using the qiagen RNeasy kit. Elute two times with 50 µl RNase free water each time

Speed vacuum to concentrate (for example until 30 µl are left)

Quantify using the nanodrop

Amplified RNA labeling for microarray

1. Reverse transcription reactions Do not forget: Use the random hexamer primers instead of the oligo-dT primer

Amplified RNA 3.475 µg Random primers 1µg/µl 1 µl H2O Up to 15 µl

Incubate for 10 min at 70 C, chill immediately on ice.

Add 10 µl of following mixture (final volume = 25 µl) 5 X First strand Buffer (contain 5 µl DTT)* Amino allyl dNTPs 10 mM 1.25 µl (dATP 10 mM, dGTP 10 mM, dCTP 10 mM, dTTP 3.33 mM, amino allyl dUTP 6.66 mM) MMLV (200 units/µl) 1 µl RNase inhibitor 0.5 µl H2O 2.25 µl * some buffer do not contain DTT, in this case add DTT to 10 mM final concentration

Mix gently by pipetting up and down, and incubate at 37 C for 1 hour. Hydrolysis Add 10 µl of 1 M NaOH and 10 µl of 0.5M EDTA pH8 Incubate at 65 C for 15 min Add 25 µl 1M HEPES pH 7.4 to neutralize the reaction

Purification Purify the amino-allyl cDNAs using the qiagen qiaqick kit, elute in two times 30 to 50 µl H2O pH8

Speed vacuum to dry

Cy3 and Cy5 labeling To the amino-allyl cDNAs (dry pellet), add 9 µl of 0.1M sodium bicarbonate. Let the cDNAs resuspend for 15 min at RT.

Add 5 µl of Cy3 or Cy5, incubate at RT for one hour.

Add 4.5 µl 4M hydroxylamine, incubate at RT for 15 min.

Combine Cy3 and Cy5 cDNAs of the couple of samples to be hybridized together on the same slide.

Clean up using the qiagen qiaquick PCR purification kit, elute two times, each time with 50 µl H2O.

Add 3 µl polyA (10 mg/ml)

Speed vacuum to dry.

Hybridization Resuspend the Cy3 or Cy5 labeled cDNAs in 40 to x µl of hybridization buffer (50% deoinized formamide, 5XSSC, 0.1% SDS) (the volume added depends on the coverslip specificities).

Incubate for 2 min at 94 C, chill on ice few min, than spin the tubes.

Hybridize overnight at 42 C.

Post-Processing of the poly-lysine slides see the protocol below: FGL Post Processing of Long Oligo Microarray Slides (Vivian Peng) revised 7/21/2005 1. No hydration of spots is necessary. 2. Cross-link 200mJ 3. Block in BSA as per Erle Lab protocol. All stock reagents should be filtered. Shake slides in 0.2% SDS Wash in copious amounts of ddH2O 4. Put slides directly into prehybridization solution [0.1% SDS, 5X SSC, 1% BSA] Pre-warm the BSA at 42oC before making the prehyb for 15 minutes. 5. Incubate at 42oC for 30-45 minutes. 6. Wash slides in many (about 5-6) changes of water. 7. Spin to dry, 1000rpm for 3 min or 500 rpm 5 mins. Notes: Plunge slides gently 5-6 times whenever transferring into new solution.