Bulk RNA extraction from low numbers of FACS-sorted cells (Version 2.0)

Version

2.0

Notice

This page is the corresponding protocol tomestone page generated as part of the ATLAS-D2K shutdown in July 2025. Many links on this page may be broken.

Authors

Michelle Southard-smith

Keywords

[‘RNA isolation’, ‘FACS’]

Subjects

[‘Gene expression analysis’, ‘Isolation, purification and separation’, ‘Molecular biology’]

Release Date

2021-07-19

Abstract

This is an protocol for isolation of RNA from Fluorescence Activated Cell Sorting (FACS) that has been optimized to improve RNA yield and implement on column DNA digestion that avoids DNA contamination of subsequent RNA samples.

Reagents

Supplies:

• RNeasy Micro Kit (with on-column DNAse I digestion kit… this is essential) (QIAGEN Cat #74004)
• RNAse-free water (Ambion, Cat# AM9932)
• Low-bind 1.5-mL RNAse-free tubes (one for each sample being collected) (USA Sci. #1415-2600)
• 1.5-mL RNase-free tubes for bioanalyzer analysis (Ambion, Cat # AM 12400)
• β-mercaptoethanol (Sigma, Cat #M3148 25ML)
• Ethyl Alcohol, 200 Proof, Absolute anhydrous ACS/USP Grade (Pharmco, Cat# 111000200)
• MultiMax RNAse-free tips
  • 10 uL tips (Cat# P-3243-10ELX)
  • 20 uL tips (Cat# P-3243-30X)
  • 200 uL tips (Cat# P-3243-200X)
  • 1250 uL tips (Cat#P-3243-1250)
• RNAse-ZAP (Sigma, Cat #R2020-250ML)
• KimTech delicate task wipers, 14.7 x 16.6 in (Kimberly-Clark, Cat# 34256)
• KimTech delicate task wipers, 4.4 x 8.4 in (Kimberly-Clark, Cat# 34155)

Procedure

Preparation for flow:

1. Prepare RNA lysis buffer
   • Add 5-10 mL RLT lysis from the kit to a 15-mL conical tube [using RNAse-free technique]
   • Then, add 10 μL β-ME per 1 mL Buffer RLT to the tube and invert to mix. This solution is stable at room temp for up to 1 month.
2. Aliquot 300 μL of RNA lysis buffer into your pre-labeled 1.5-mL collection tubes for FACS

I. Procedure for the day of flow:

1. After collecting cells at the flow core, invert the tube several times to ensure all cells are exposed to lysis buffer.
2. Leave collected samples on ice for the duration of the sort.
3. Back in lab, vortex the samples intermittently for 5-10 seconds over the course of 30 minutes to ensure complete lysis. Samples should be left on ice during this time.
4. Samples can be stored at -80oC for >1 month without any noticeable reduction in RNA quality.

II. Preparation for RNA extraction

1. Prepare DNAse I on-column digestion solution as per instructions (QIAGEN, comes with the kit)
   • Note: DNAse I solution (and thawed aliquots) are stable for 6 weeks at 4C  You’ll use 10 μL for each reaction, so estimate your use accordingly
2. Prepare ~40 mL of 70% EtOH and 80% EtOH solutions
   • Use Ambion RNase-free water
3. Ensure that 100% ethanol has been added to Buffer RPE (as indicated on the bottle)
4. Remove RNA columns for the kit from the refrigerator and label them with your sample IDs
5. Label 1.5-mL elution tubes with complete sample information for long-term storage
6. Pre-label 1.5mL RNAse-free Ambion tubes with the Core project # and sample id
   • i.e. 364-YourInitials-1, 364-YourInitials-2, etc. (according to core instructions)
7. Prepare on-column DNAse I digestion solution master mix:
   • Add 10 μL DNAse I stock to 70 μL Buffer RDD from the refrigerated kit
   • Keep on ice until ready to use
   • NOTE: If have >3 samples, make a Master Mix of DNAse I solution:
   • Calculate the # of reactions you need with an extra 0.3X reaction e.g. 7 + 0.3= 7.3X
   • Combine the appropriate amts of Buffer RDD and DNAse I
   • Gently invert tube to mix and then briefly centrifuge ( Do NOT vortex DNAse I)

III. RNA extraction protocol

1. Disinfect bench with bleach if you’ve been working with DNA or if it needs a “deep clean”
2. Spray RNAse-ZAP onto a large kim wipe and wipe off bench, tube racks, pipette tip boxes, pipettors, centrifuges, etc. that you’ll be using.
3. Fill up 1-2 ice buckets depending on the number of samples you’re extracting
4. Place a metal tube rack on the ice (and wipe with RNAse-ZAP)
5. If RNA lysates were frozen, place tubes in 37oC water bath until just before finished thawing
   • Avoid making contact of the lids with the water! If so, carefully wipe off with RNAse-ZAP and centrifuge tubes briefly before opening them
   • Leave tubes at room temp from this point on (unless you can’t start right away)
6. Add 1:1 volume of 70% EtOH solution to your sample tube (ALL PERFORMED AT ROOM TEMP) and immediately pipette up/down to gently mix the RNA lysates with the ethanol
7. Transfer 400-500 μL of this solution to the respective pre-labeled RNA column.
   • Avoid over-filling, which can make a mess. Save remaining samples in their tubes.
8. Repeat the previous 3 steps for the rest of your samples
   • If you have a lot of samples, you’ll notice a majority of the solution will have “leaked” through the column- this is fine.
9. Centrifuge loaded RNA columns at 10 xg for 5 mins
   • If you are having problems getting a good yield in your samples, one way to combat this is to reapply the flow through to the column and respin at 10 xg for 5 minutes. You can do this 2-3 times to achieve maximum results.
10. Add remaining samples to the RNA columns and fill up the column to the 500 μL mark with the flow-through at the bottom of the collection tube
11. *Discard all remaining lysate in the collection tube, or it might overflow!!
    • This lysate contains β-ME, so it should not be thrown in the trash due to the smell
12. Centrifuge at 10 xg for 5 mins ; then centrifuge 15 s at 13,000 xg before proceeding
    • NOTE: If you still have remaining sample to load, repeat the previous 3 steps as necessary before cranking up the speed on the centrifuge
13. Replace the collection tubes on the column before proceeding!
    • Discard used lysate from old ones in the β-ME waste beaker NOW FOLLOW THE RNeasy Micro Handbook PROTOCOL (with slight modifications):
14. Add 350μL Buffer RW1 to the RNeasy MinElute spin column. When adding the Buffer RW1, slowly dispense the liquid in a circular motion (i.e. clockwise or CCW) around the tube to rinse the inside wall of tube
15. Centrifuge 15seconds at 10,000 xg
16. Remove all remaining buffer RW1 from the O-ring in the column using a 10μL pipette tip
17. Apply 80μL of DNAse I Master Mix (prepared ahead of time) directly onto the dried columns
    • Avoid puncturing the column with the pipette tip. Cover the entire O-ring by gently flicking the tube to eradicate all DNA
18. Incubate at RT on the benchtop for 15 mins.
    • After about half the time has elapsed, pick the spin column up and move it in a quick circular motion (mimicking the movement of a centrifuge)
19. Add 350 μL Buffer BW1 to the MinElute spin column
20. Centrifuge 15s at 10,000 xg; Replace collection tubes and discard the used ones
21. Add 500 μL Buffer RPE to the column
22. Centrifuge 15s at 10,000 xg; Replace collection tubes and discard the used ones
23. Add 500 μL of 80% EtOH to the column
24. Centrifuge 2 min at 10,000 xg; Replace collection tubes and discard the used ones
25. Place tubes in the centrifuge and open the lid to the MinElute column
26. Centrifuge 5 min at > 12,000 xg (or full speed). Slowly increase the centrifuge speed. Start around 1,000 xg and double the speed each time until you reach >12,000 xg (i.e. start at 1,000 xg  2,000 xg  4,000 xg  8,00xg  >12,000 xg).
27. Transfer the column onto a pre-labeled 1.5 mL sample collection tube
28. Add 14 μL RNase-free water directly to the center of the spin column membrane and let sit for 1 min to absorb. Add the water one drop at a time with the intention of maximally saturating the membrane.
29. Centrifuge for 1 minute at >10,000 xg. Slowly increase the centrifuge speed (reference Step #27)
30. Re-apply the flow-through to the column membrane
31. Rotate the column the OPPOSITE direction from its original position in the centrifuge
32. Centrifuge for 1 minute at >10,000 xg
    • Start off at ~1600 xg, gradually increase the speed to ~2,600 xg, (this helps prevent caps from ripping off of the tubes and may also help more RNA be eluted from the column)
33. Place samples in metal ice block after confirming the sample eluted properly and that the column and tube labels correspond
34. Aliquot 0.5 to 2 μL of sample into prelabeled tubes in metal ice block for sending to QC analysis.
35. Freeze samples at -80oC for long-term storage

Consortium

GenitoUrinary Development Molecular Anatomy Project (GUDMAP) Consortium