Quantitative polymerase chain reaction (qPCR)

When working with RNA it is very important to work in an environment that is free of RNAses. Simple precautions such as having reserved pipettes for use only with RNA procedures and spraying the work area with a decontaminant reagent (e.g., RNAse Away) before beginning the procedure are very helpful.

Total RNA extraction of cells from FACS using TRIzol Reagent and TRIzol LS Reagent

1. After collecting the sorted cells in a 1.5 ml centrifuge tube*, add TRIzol LS Reagent (3 volume of the sorted cells) immediately. TRIzol reagent contains phenol and should only be used under a fume hood.
   *Option: sort cells directly into TRIzol Reagent (if the volume is small) or TRIzol LS Reagent (if the volume is large; adjust final volume for 3:1 Trizol LS:cell ratio)
2. Lyse the cells by pipetting several times, and add TRIzol reagent to a total volume of 1 ml. Add 10 µg glycogen as a carrier to assist RNA precipitation. Gently shaking samples for 1 minute to help lysis completely. At this stage, the sample can be flash frozen in liquid nitrogen and stored at -80 °C or the protocol can be continued.
3. To continue add 0.2 ml of chloroform. Shake hard by hand for at least 15 seconds (do NOT vortex). The sample should be light pink.
4. Incubate the sample for 2 minutes at room temperature and then centrifuge at 12,000 x g for 15 minutes at 4 °C.
5. The mixture will separate into a lower red phenol-chloroform phase, an interphase, and a colorless upper aqueous phase. The upper aqueous phase contains the RNA. The top layer should comprise of about 60% of the initial volume of TRIzol (approximately 0.6 ml). Transfer 0.5 ml of the top aqueous layer into a new RNAse-free 1.5 ml microfuge tube by using a 200 μl pipette being careful not to transfer any of the interphase layer.
6. To precipitate the RNA add 0.5 ml isopropanol.
7. Allow the sample to sit at room temperature for 10 minutes and then centrifuge the sample at 12,000 x g for 10 minutes at 4°C. RNA will form a gel-like pellet on the bottom of the tube.
8. Without disturbing the pellet, remove the supernatant with a pipette and wash the pellet in 1 ml 75% ethanol. Mix the sample by gentle inversion and centrifuge at 7,500 x g for 5 minutes at 4°C.
9. After centrifugation, remove the ethanol with a pipette and allow sample to dry while inverted for 10 minutes.
10. Resuspend the pellet in 10-20 μl of RNAse-free water and incubate the sample at 55°C for 10 minutes. It is recommended to finger vortex frequently during incubation to aid in RNA rehydration. Optionally, you can check sample quality and quantity using a NanoDrop.

Materials

Invitrogen™

TRIzol™ Reagent                                          Cat no. 15596026

TRIzol™ LS Reagent                                     Cat no. 10296010

GlycoBlue™ Coprecipitant (15 mg/mL)          Cat no. AM9516

Reference

Peterson SM, Freeman JL. RNA isolation from embryonic zebrafish and cDNA synthesis for gene expression analysis. J Vis Exp. 2009 Aug 7;(30):1470. doi: 10.3791/1470. PMID: 19684565; PMCID: PMC3152201.

RNA extraction protocol, Version 1.8 (4/2020). Transcriptional Regulation & Expression Facility. Ithaca, New York