RNA extraction

Reagents required:

RNase Zap (Sigma R2020-250ML)

1ml, 200ul, 20ul and 10ul Rnase free tips and the corresponding pipettes Rnase free tubes (Eppendorf biopur 1.5ml tubes)

Trizol reagent (Invitrogen 15596-026) 2ml heavy phase lock tubes from 5prime (yellow tube) Chloroform (Sigma 2432)

Isopropanol ( grade I9516-500ML ) Glycogen (Invitrogen 10814-010)

Ethanol (molecular biology grade E7023-500ML )

Nuclease free water (Qiagen 129114 ) Rnase out (Invitrogen 10777019)

1. Samples for extraction should be stored frozen in Trizol reagent (Invitrogen). The maximum sample volume should not be more than 600ul if using a heavy phase lock tube at the later stage of extraction. 2. Remove the sample from -80C and thaw on ice. 3. Top up the sample volume with Trizol to 500ul. (Blueprint samples are normally frozen down in 200ul trizol). 4. Vortex the sample for 30 seconds to insure complete has occurred, then allow the sample to warm up to room temperature, Incubate the homogenized sample (see Homogenizing samples ) for 5 minutes at room temperature to permit complete dissociation of the nucleoprotein complex. 5. Spin down the 2ml heavy phase lock tubes for 30sec at 12000g. This is to pellet the gel to the bottom of the tubes) 6. Transfer the 500ul of sample to a 2ml heavy phase lock tube (5prime).

7. Add 0.2 mL of chloroform per 1 mL of TRIzol® Reagent used for homogenization. Cap the tube securely. 8. Shake tube vigorously by hand for 15 seconds. 9. Incubate for 2–3 minutes at room temperature. 10. Centrifuge the sample at 12,000 × g for 15 minutes at 4°C. 11. Note: The mixture separates into a lower red phenol-chloroform phase, an interphase, and a colorless upper aqueous phase. RNA remains exclusively in the aqueous phase. The upper aqueous phase is ~50% of the total volume.

12. Remove the aqueous phase of the sample (and KEEP) by angling the tube at 45° and pipetting the solution out. Avoid drawing any of the interphase or organic layer into the pipette when removing the aqueous phase. The phase lock gel should have formed a complete barrier between the upper aq layer and the phenol chloroform phase. 13. Place the aqueous phase into a new tube and proceed to the RNA Isolation Procedure.

RNA isolation

RNA precipitation 1. When precipitating RNA from small sample quantities (<106 cells or <10 mg tissue), add 5–10 µg of RNase-free glycogen as a carrier to the aqueous phase. I always add 10ug of glycogen which is equal to 0.5ul of the 20ug/ul stock (Invitrogen 10814-010). The glycogen should be aliquoted into single use aliquots and stored at -20C. 2. Note : Glycogen is co-precipitated with the RNA, but does not inhibit first-strand synthesis at concentrations ≤4 mg/mL, and does not inhibit PCR. 3. Add 0.5 mL of 100% room temperature isopropanol to the aqueous phase, per 1 mL of TRIzol® Reagent used for homogenization. 4. Incubate at room temperature for 10 minutes. 5. Centrifuge at 12,000 × g for 10 minutes at 4°C. 6. Note: The RNA is often invisible prior to centrifugation, and forms a gel-like pellet on the side and bottom of the tube. 7. Proceed to RNA wash .

RNA wash 1. Remove the supernatant from the tube, leaving only the RNA pellet. This is easiest if you only remove one tube from the rotor at a time, so that the pellets do not slip down the sides of the tube. Remove as much isopropanol as possible. 2. Wash the pellet, with 1 mL of ice cold (put at -20C before use) 75% ethanol per 1 mL of TRIzol® Reagent used in the initial homogenization. 3. Note : The RNA can be stored in 75% ethanol at least 1 year at –20°C, or at least 1 week at 4°C.

4. Add the ethanol to the RNA pellet and gently flick the tube to loosen the pellet, but without breaking it up too much, DO NOT VORTEX. Then centrifuge the tube at 8000 × g for 10 minutes at 4°C. Discard the wash. 5. Repeat the wash step described above, after the second wash remove all traces of ethanol from the tube. Small volumes can easily be removed with a P10. Leave the tube open. 6. 4. Air dry the RNA pellet for 5–10 minutes. Do not dry the pellet by vacuum centrifuge. 7. Note: Do not allow the RNA to dry completely, because the pellet can lose solubility. Partially dissolved RNA samples have an A260/280 ratio <1.6. 8. 5. Proceed to RNA resuspension .

RNA resuspension 1. Resuspend the RNA pellet in RNase-free water (20–50 µL) by passing the solution up and down several times through a pipette tip. For microarray analysis from 2e6 cells I usually resuspend in 30ul of nuclease free water. 2. Once the pellets have been resuspended place on ice.

RNA quality control

Remove a 3ul sample into a separate Rnase free tube for nanodrop and agilent analysis. Take a 1ul sample from the 3ul sample and read the RNA concentration on the nanodrop. Freeze the remaining 2ul of RNA at -80C ready to run on the agilent.

QC

Remove the 2ul samples from -80C and denature at 70C for 2 minutes. Then cool the samples on ice. Run the samples on the agilent as per the protocol. To run on the array the RNA integrity number (RIN) should be 7 or above.

RNA QC

Nanodrop

RNA absorbs maximally at 260nM. The ratio of absorbance at 260nM to 280nM is used to assess the purity of the RNA. Pure RNA has an absorbance ratio of 2.1. The purity value obtained can be pH dependant, as some of the contaminants like have an absorbance that is pH dependant (so RNA should not necessarily be measured on the nanodrop in pure water).

Any peak observed on the nanodrop at 230nm is NOT RNA but contamination.

Three sources of contamination produce peaks in the 230nm range:

Proteins, chaotropic salts and phenol

So the 260/230 ratio should also be close to 2.

Isopropanol/ethanol contamination would cause the 260:280 ratio to drop below 2 if the sample was measured in pure water.

Guanidine isothiocyanate- this hardly influences the A260:A280 ratio, so has a small effect on RNA quantification. BUT is causes the A260:A230 ratio to drop significantly. An A260:A230 ratio below one should be avoided.

Phenol- has a very strong effect on the quantification of RNA- it can cause an overestimation of the amount of RNA present. There is a contaminating absorbance peak at 270nM, which IS NOT RNA.

Agilent data- typical trace showing intact RNA

Marker peak

- Depending on the RNA extraction method, the small 5S, 5.8S and tRNAs may be present in the electropherogram between the marker peak and the 200 nucleotide region.

- Two ribosomal peaks, the 18S and 28S rRNA peaks

- The baseline between the internal marker and the 18S rRNA peak is relatively flat and free of small rounded peaks corresponding to smaller RNA molecules that are degradation products of the rRNA transcripts

In general, when analyzing good quality RNA, the height of the 28S rRNA peak should be at least twice that of the 18S rRNA peak. For perfect quality RNA we indeed find ratios over 2 with the Bioanalyzer. However, almost exclusively RNA isolated from samples results in 28S/18S ratios larger than 2.