RNA extraction using Qiazol
Take the normal(N1-N2) and tumor tissues’ powders (T1-T2) from -80°C(20-30mg)
- Add 1 mL of Qiazol Reagent to lyze samples and homogenize by pipetting up and down. Incubate for 5min to permit complete dissociation of the nucleoproteins complex. (Qiazol vs Trizol: QIAzol is optimized to lysis tissues that have a high content of fats)
- Add 0.2 mL of chloroform per 1 mL of Qiazol Reagent used for lysis, then securely cap the tube. Incubate for 2–3min. Centrifuge the sample for 15min at 12,000 × g at 4°C. The mixture separates into a lower red phenol-chloroform, and interphase, and a colorless upper aqueous phase. Transfer the aqueous phase containing the RNA to a new tube by angling the tube at 45° and pipetting the solution out.
- Add 0.5 mL of isopropanol to the aqueous phase, per 1 mL of Qiazol Reagent used for lysis. Incubate for 10min. Centrifuge for 10min at 12,000 × g at 4°C.
- Discard the supernatant and resuspend the pellet in 1 mL of 75% ethanol per 1 mL of Qiazol Reagent used for lysis. Vortex briefly, centrifuge for 5 min at 7500 × g at 4°C. Discard the supernatant and vacuum or air dry the RNA pellet for 5–10 min. Resuspend the pellet in 20–50 μL of RNase-free water. Incubate in a water bath or heat block set at 55–60°C for 10–15 min. Determine the RNA yield and purity using Nanodrop.
Calculate the RNA concentration using the formula A260 × dilution × 40 = μg RNA/mL. Calculate the A260/A280 ratio. A ratio of ~2 is considered pure N1(1277 ng/ul), T1(815 ng/ul), N2(1557 ng/ul), T2(1616 ng/ul)
RT PCR
- Program the thermal cycler so that cDNA synthesis is followed immediately by PCR amplification, as follows: cDNA synthesis and pre-denaturation, 1X, 55°C for 30 min, 94°C for 2 min PCR: 40X cycle, 94°C for 15 seconds, 60°C for 30 seconds, 68°C for 4 min(1min/kb) Final extention (optional): 1X, 68°C for 5 min Add the following to a thin-walled PCR tube on ice. 2X Reaction Mix(12.5μL), Template RNA (1ug), Sense primer,10 μM (0.5 μL), Anti-sense primer,10 μM(0.5 μL), SuperScriptTM III RT/PlatinumTM Taq Mix (0.5μL), Autoclaved distilled water(Till 25 μL) Primer pairs: DNAJB1 ex1& PRKACA ex3 , GAPDH (control)
- Verify the absence of genomic DNA in RNA preparations using One taq master mix plus GAPDH primer pairs and RNA (NO RT control).
- Gently mix and make sure that all the components are at the bottom of the tube. Centrifuge briefly. Place the reaction in the preheated thermal cycler programmed as described above. Load it on the 1% agarose gel and analyze the results.
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