RNA extraction and quantitative real-time PCR (qPCR)

Dear Katja,

Thank you for reaching out. We have attached the protocols for RNA purification, DNAse treatment, Reverse transcription and qPCR.

Please feel free to let us know if we can be of further assistance.

Best regards,

Ziru & Ormond

RNA Purification Protocol

1. Prepare bench for RNA purification:
   a. Spray bench & pipettes with 70% ethanol and RNAse-away
   b. Obtain new filter tips for pipettes and Eppendorf tubes that are RNAse & DNAse free

2. Prepare sample:
   a. Tissue: Place frozen tissue (adipose, liver, etc.) on heavy duty foil folded over twice (so the tissue is wrapped in two layers of foil)
      i. Prepare dry ice and a pre-chilled metal block in the dry ice and liquid nitrogen.
      ii. Dip wrapped tissue in foil into liquid nitrogen until the humming noise disappears, put the wrapped tissue on the metal block surrounded by dry ice, immediately use a hammer to break the tissue by hammering ~10 times
      iii. Repeat submerging the wrapped tissue in liquid nitrogen and hammering at least twice (three times on some tough tissues, e.g. sWAT, tail, skin, muscle etc.)
      iv. After the second round of hammering, re-submerge the tissue in liquid nitrogen, which refreezes the tissue into a powder. Transfer the powder into pre-cooled tubes ASAP. 
      v. Tips: disperse the powdered tissue into the tube by tapping the foil or use pre-chilled tools (e.g. spatula or forceps) before transferring into tubes
      vi. Add 1ml RNA-STAT 60 (stored at 4°C) and vortex vigorously for 10 seconds. Big chunks of tissue may still be visible. Put the sample on ice and it is ready for future homogenizing.
         1. STAT 60 is phenol-based and is toxic; use gloves AND goggles when handling/homogenizing tissue. Tissue can also be homogenized in the fume hood to reduce breathing vapor
         2. If using 1.5 mL Eppendorf tubes, adding less RNA-Stat 60 for the homogenization step (200-500 uL) will reduce spillage; add up to 1 mL RNA STAT60 total after homogenization

   b. Floated adipocytes/SVF or cultured cells:  
       i. Cells do not need to be hammered. They will dissociate with homogenization. Discard medium. Add 1 mL RNA-STAT60 per cell culture well , vortex vigorously, and proceed with step 3 (alternatively, add less STAT60 initially to reduce spillage and add up to 1 mL after homogenization)
         1. Alternatively: a needle + syringe can be used to break up cells... uptake and expel the cell-liquid mixture vigorously to break up cells

3. Wash the homogenizer tips with water and pre-spray with 70% ethanol and RNAse-away; homogenize each sample and change out tips between samples. Keep tubes (with STAT 60 and tissue) on ice during homogenization. Generally, 10-20 seconds per tissue should be enough. Wait until big chunks of tissue disappear
   a. Tip: Keep tissues cold! The homogenization process will generate heat

4. 1st centrifuge: 14,000 rcf, 15 min at 4 ̊C

5. Transfer the clear supernatant into a new tube and get rid of the tissue debris

6. Leave the tube at room temperature for 5-10 min before adding chloroform, because sometimes if we add the chloroform to the STAT 60 at cool temperature, it will generate pink water phase for the next step (not sure why)

7. Add 200uL chloroform (stored at RT) to the tube, invert for 30-40 times to mix the solution well. Leave at room temperature for 5 min

8. 2^nd centrifuge: 14,000 rcf, 15 min at 4 ̊C      
   a. Set up new 1.5 ml Eppendorf tubes and pre-cool on ice

9. Transfer the top water phase into the new pre-cooled tube. 
   a. Tip: transfer quality is more important than quantity. Don’t touch the middle white layer, leaving some volume above the white layer is okay

10. Add an equal volume of isopropanol into the tube, invert the tube 30-40 times to mix well

11. Put the tubes into the -20C freezer overnight

12. 3rd centrifuge: 14,000 rcf, 15 min at  4 ̊C      

13. Remove all the supernatant, which contains isopropanol
   a. Tip: try your best to remove all the isopropanol by pipetting, because remaining isopropanol will cause contamination in your RNA sample, visible via Nanodrop

14. Wash the pellet with 75% ethanol (use DEPC/Ultrapure water), the pellet should dislodge from the bottom of the tube and be free floating. Invert the tube sharply to wash the pellet or tap on the tube lightly to dislodge the pellet

15. 4^th centrifuge: 14,000 rcf, 5 min at 4 ̊C      
   a. For improved RNA quality, repeat steps 14-15 two more times (for a total of three washes)

16. Remove all the supernatant from the tube and leave only the pellet behind. Here, really try to remove as much as possible as we need to dry the pellet

17. Dry the pellet: leave the lids open for 10 min and keep on ice OR leave lid open and put tube on 55°C heat block for 5-10 seconds at a time until all liquid evaporates

18. When the pellet looks half-transparent, dissolve the RNA in water (use Ultrapure water), water amount varies by the size of pellets (the range is usually between 10-50 uL water)

19. Mix well by pipetting up and down and the sample is ready for the Nanodrop: to assess quality and concentration 
20. add DNAse treatment

DNAse treatment

The purpose of DNAse treatment is to remove DNA contamination from RNA samples. However, this step could be ignored if you included an intron in your primer design.

The kit used is DNA-free™ DNA Removal Kit Catalog number: AM1906.

After determining RNA concentration and quality (A260/A280) ideally over 1.7- in nanodrop:

• Mix RNA and H₂O to make all samples 200 ng/µl, 30 µl total volume (or 50 µl if you have enough RNA; 1 µl of DNAse could digest 1-10 µg). Example: 

• Add 3.4 µl 10X DNAse buffer per tube, then 1 µl DNAse per tube (mix, or mix the buffer and the DNAse together first) (DON´T bring DNAse to the bench on ice. It’s an enzyme, so bring it in the blue box (-20C)
• Incubate for 30 minutes at 37°C
• Add 3.8 µl DNAse inhibitor to stop reaction (Don't use pipette tip whose hole is too small (i.e.  p10),  you might get more liquid from DNase inhibitor, not equal proportion of liquid and resin)
• Mix tubes and incubate at RT 2 minutes (mix occasionally because resin and liquid separate easily)
• Spin down at 10,000 x g for 1.5 minutes, transfer to a new tube
• Nanodrop samples again (concentrations will be lower after DNAse treatment)
   

Reverse Transcription Protocol

• Dilute RNA with H₂O in order to have 1 µg RNA in 10 µl. 
  Example: 

(Can use 1 ng - 5 µg of RNA per 20 µl reaction)

2. Heat mixture to 65°C for 5 minutes on a thermocycler. Then chill samples on ice. 

Use a table-top centrifuge to pull sample to bottom of tube.

Invitrogen™ M-MLV Reverse Transcriptase (200 U/µL) (Cat # 28025013)

*These two are enzymes, so bring them to the bench into the blue box (-20°C), NOT ON ICE

RT protocol: 

1. 25°C for 10 minutes

2. 37°C for 50 minutes

3. 70°C for 15 minutes

qPCR

1. Protocol with dilution of your cDNA

  ● By the end of RT you have 20uL of cDNA, add 80 µl dH2O, (1/5 dilution). This is the stock.
  ● Mix well, take 50 µl stock. Add 100 µl dH2O (1/3 dilution).

2. Protocol without dilution of your cDNA 
 

Run plate on qPCR thermal cycler

*Run samples in duplicate
*Run a calibration curve for each gene you test with serial dilutions of a sample where you know your gene is highly expressed (example of calibration curve in the protocol for Primer Validation)

You always have to run qPCR of reference genes (PPIA, GAPDH, RpL32, HPRT, TBP etc.) in parallel with your target gene.

qPCR data Analysis

● Calculate Log of the ratio of dilution curve (these values will be on your x axis)
● Take average of CT for each dilution (these values will be on your y axis)
● Calculate intercept, slope, RSQ
● Repeat for reference gene
● Calculate Adjusted CT of your samples (apply the equation of the line to your samples CT)
● Take the Log of CT
● Repeat for reference gene and calculate geomean of multiple reference genes.
● Divide the Log (gene in sample X) by Log (reference gene in sample X)
● Normalize dividing each value by the average obtained in the controls.
● Average duplicates
● Get average and SD of biological replicates