Reverse transcription (RT) and quantitative RT-PCR analysis

RNA extraction

1. Wash cells in the culture plate with 1xPBS for 1-2 times after removing the medium.
2. Add 1ml TriZol per 10 cm dish and pass the cells several times through tips to homogenize.
3. Transfer the lysates to RNase-free Eppendorf tubes and incubate the samples for 5-10 mins at RT.
4. Add 200 ml chloroform and shake the tubes vigorously for 15 seconds, followed by 2-3 mins incubation at RT.
5. Spin the samples at 12,000 g for 15 mins at 4°C in a microcentrifuge.
6. Transfer the upper clear aqueous phase to new RNase-free tubes and precipitate the RNA by adding 0.5 ml of isopropanol.
7. Incubate the samples for 10 mins at RT and spin at 12,000 g for 10 mins at 4°C in a microcentrifuge.
8. Remove supernatant and wash the pellets at least once with 1 ml 75% ethanol (pre-cold) in RNase-free water, vortex and centrifuge at 75,00 g for 5 mins at 4°C.
9. Remove ethanol and briefly air-dry the pellets for 5-10 mins.
10. Dissolve RNA in RNase-free water.
11. Determine RNA concentrations using NanoDrop.

Reverse transcription

1. Mix the following components into a microcentrifuge tube to the total volume of 15 ml:   
   1. 0.5 or 1 mg total RNA           
   2. 0.5 mg poly-(T)20 primer
   3. RNase-free water
2. Heat the tube to 70°C for 5 mins and cool the tube immediately on ice.
3. Add the following components into the tube:
   1. M-MLV 5x Reaction buffer                                     5 ml
   2. dATP, 10 mM                                                          1.25 ml
   3. dCTP, 10 mM                                                          1.25 ml
   4. dGTP, 10 mM                                                          1.25 ml
   5. dTTP, 10 mM                                                          1.25 ml
   6. Recombinant RNasinâ Ribonuclease Inhibitor      25 units
   7. M-MLV RT                                                              200 units
   8. Nuclease-free water to the final volume               25 ml
4. Mix the solution gently by flicking the tube and incubate at 37°C for 60 mins and cool the product at 4°C for 10 mins.

RT-qPCR

1. The following components are mixed for one reaction:
   2x Sybr Green Supermix              10 ml
   Forward primer, 10 mM              1.5 ml
   Reverse primer, 10 mM               1.5 ml
   cDNA                                           100 ng
   Nuclease-free water                   Variable
   Total volume                               20 ml
2. Load and seal the plate with adhesive film, and spin the plate briefly.
3. Load the plate into BioRadâ CFX96ä Touch, and run the PCR using the following program:
   95°C          30 sec
   95°C          15 sec   40 cycles
   60°C          30 sec    
   65-95°C, 0.5°C increments at 5 sec/step (Melt Curve)
4. Analyze the data using ∆∆CT method.

The primers used in these studies include:

SPIN1, 5’-CAGAGCTGATGCAGGCCAT and 5’-ACTGGGTAACAGGGCCATTG 

p53, 5’-CCCAAGCAATGGATGATTTGA and 5’-GGCATTCTGGGAGCTTCATCT 

p21, 5’-CTGGACTGTTTTCTCTCGGCTC and 5’-TGTATATTCAGCATTGTGGGAGGA 

PUMA, 5’-ACAGTACGAGCGGCGGAGACAA and 5’-GGCGGGTGCAGGCACCTAATT

pre-rRNA, 5’-GCTCTACCTTACCTACCTGG and 5’-TGAGCCATTCGCAGTTTCAC

18S rRNA, 5’-GCTTAATTTGACTCAACACGGGC and 5’-AGCTATCAATCTGTCAATCCTGTC

rRNA, 5’-TGAGAAGACGGTCGAACTTG and 5’-TCCGGGCTCCGTTAATGATC

GAPDH, 5’-GATTCCACCCATGGCAAATTC and 5’-AGCATCGCCCCACTTGATT

(Fang Z et al. eLIFE. 2018)

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