RNA extraction

From Molecular Biology Wiki

RNA makes up transfer RNA, ribosomal RNA, messenger RNA and the most recently-discovered short inverted RNA (or silencer RNA). The messenger RNA is considered the transcriptome. And assaying for expression levels show that cells can upregulate and downregulate expression of most proteins via the amount of gene-specific mRNA.

In order to measure the expression level of mRNA via PCR, one must extract the RNA, quantitate total RNA (if possible), purify RNA by digesting any residual DNA that is commonly co-extracted with RNA, reverse transcribe the RNA into complimentary DNA using the enzyme Reverse Transcriptase, then use PCR to amplify the cDNA to quantifiable levels.

RNA extraction can be accomplished by many methods and RNA extraction kits are very common, but most of those kits include an RNA size limit where RNA less than 250 bp is less likely to be retained. The following is the most common RNA isolation method adapted for our lab.

Materials Needed for Trizol LS RNA Extraction

• Trizol LS (store at 4 C)
• Chloroform (store at 4 C)
• Isopropanol
• 75% Ethanol
• Molecular Grade Water (Dnase, Rnase, Pyrogen Free)
• 1X PBS
• Microcentrifuge

Procedure of Trizol LS RNA Extraction

Sample Preparation

1. If sample is tissue it is important to homogenize the tissue in a buffer such as PBS use a ratio like 350mg tissue to 1ml PBS.
2. If sample is liquid like conditioned media you may want to dilute it with PBS.

Trizol LS RNA Extraction

1. Pipet 300µl homogenized sample into clean epi tube.
2. Add 800µl Trizol LS. Mix by inverting.
3. Incubate 10 min at RT.
4. Add 0.2ml Chloroform. Mix vigorously by hand.
5. Incubate 15 min at RT.
6. Centrifuge 12,000g for 15 min, RT.
7. Transfer upper aqueous layer to a clean 1.5 ml epi tube.
8. Add 600µl Isopropanol to tube and invert several times. Incubate 10min, RT.
9. Centrifuge at 12,000 g for 10 min.
10. Pour off and discard supernatant. Wash pellet with 500µl 75% Ethanol.
11. Centrifuge 7500g for 5 min.
12. Pour off and discard supernatant. Look for pellet. Invert tube on paper towel and air dry pellet.
13. Resuspend pellet in 20-100µl MG H2O.
14. Perform Dnase I digestion on entire amount of RNA. Label should include Pure RNA.
15. Perform OD260/280 measurements and calculate the RNA concentration.
16. Use equal amounts of starting RNA template for all Reverse Transcription reactions.

Protocol Notes

You can buy Trizol LS from Invitrogen, recombinant Dnase I from Ambion, Molecular Grade water or HPLC water from Sigma, spectrophotometer like Spectramax from Molecular Devices.

References for Trizol LS RNA Extraction

1. For more info see Invitrogen's TRIzol LS Reagent Product Sheet.