P-FPMI-109 - P-FPMI-109
RNA isolation using RNeasy Mini Kit
Qiagen: Cat # 74104; for 50 reactions; or Cat # 74106 for 250 reactions
PURPOSE: To permit isolation of very pure RNA for microarray and PCR amplification work.
The procedure written below can be used for 1.5 to 2 x 107 PMA-primed, plastic-adherent THP1cells.
„« All RNeasy protocol should be performed at room temperature (approximately 23C).
„« Buffers RLT and RW1 contain a guanidine salt and therefore are not compatible with disinfecting agents such as bleach.
„« Buffer RLT may form a precipitate. If so re-dissolve by warming at room temperature and keep at room temperature until used.
Preparation before procedure
1. Lysis buffer: Buffer RLT + beta-ME beta-mercaptoethanol). Add 10 ul beta-ME per 1ml buffer RLT before use.
2. Calculate the total volume of RLT buffer required and add appropriate amount of beta-ME.
3. Add x 4 volumes of ethanol to Buffer RPE (as indicated on the bottle of the buffer), to obtain a working solution.
4. For the on-column DNase digestion with RNase-free DNase (Qiagen cat # 79254), prepare a DNase I stock solution by dissolving solid DNase I (1500 Kunitz units) in 550 ul RNase free-H2O provided in the kit. Mix gently by inverting the tube. DO NOT VORTEX. For long term storage, make single use (40 ul / tube; for 4 reactions) aliquots and store them at -20 C for up to 9 months. DO NOT FREEZE THAW. Before use thaw and DO NOT CENTRIFUGE.
5. NOTE: there are collection tubes of two different volumes; 1.5 ml for elution and 2 ml for all other steps.
1. Remove tissue culture supernatant from the adherent cells, save the supernatant at - 20C for further analysis.
2. Wash the adherent cells with 10 ml PBS once.
3. Just before you are ready to process the cells, remove the PBS and add 1.2 ml lysis buffer and scrape the cells using a cell scrapper.
4. Pipette 600 ul cell suspension into each of two Eppendorf tubes and vortex these samples.
5. Add 600 ul of 70% ethanol to the suspension, mix well by pipetting. DO NOT CENTRIFUGE. NOTE: A visible precipitate may form after the addition of ethanol; this will not affect the isolation procedure.
6. Apply 600 ul of sample to the RNeasy mini column placed in a 2 ml collection tube supplied with the kit (including any precipitate that is formed).
7. Close the tube gently, centrifuge for 15 sec at greater than 8000 x g (greater than 10,000 rpm).
8. Discard the flow through, reuse the collection tube, apply the rest of the sample to the mini column and repeat sample loading as in step 8.
9. Pipette 350 ul buffer RW1 into the mini column and centrifuge for 15 sec at greater than 8000 x g (greater than 10,000 rpm) to wash.
10. For each sample, add 10 ul DNase 1 stock solution to 70 ul Buffer RDD (supplied in the RNase-free DNase set; Qiagen cat # 79254). This volume supplies one reaction, so you can make appropriate amount depending on the number of reactions. Mix gently by inverting the tube.
11. NOTE: DNase I is especially sensitive to physical denaturation. Mixing should be carried out by gently inverting the tube. DO NOT VORTEX.
12. Add 80 ul of the DNase I incubation mix DIRECTLY onto the silica-gel membrane of the column and place on bench top (RT) for 15 min.
13. NOTE: Make sure to pipette the DNase I incubation mix DIRECTLY onto the Silica-gel of the mini column. DNase digestion will be incomplete if part of it sticks to the walls or the O-ring of the column.
14. Pipette 350 ul Buffer RW1 into the mini column and centrifuge for 15 sec at greater than 8000 x g (greater than 10,000 rpm), discard the flow through.
15. Transfer the RNeasy column to a new 2 ml collection tube, pipette 500 ul Buffer RPE onto the column, close the tube gently and centrifuge for 15 sec at greater than 8000 x g (greater than 10,000 rpm) to wash. Discard flow thru. This wash is done X 2.
16. After the second wash, discarding the flow through each time, gently close the tube and centrifuge for 2 min at greater than 8000 x g (greater than 10,000 rpm) to dry the RNeasy silica gel membrane.
17. Place the column in a new 1.5 ml collection tube for elution.
18. Pipette 30 ul RNase-free water directly onto the silica-gel membrane. Close the tube gently and centrifuge for 1 min at greater than 8000 x g (greater than 10,000 rpm). Repeat these elution steps twice. The total sample volume eluted from the columns will be around 60 ul.
19. Pool the two identical samples together, the total volume per sample will be 120 ul.
20. Speed vac and concentrate the sample to approximately less than one-third the volume.
21. NOTE: Required concentration of total RNA for microarrays cannot be less than 1 ug/ul.
22. Add 1 ul RNase Inhibitor to your sample (NOTE this is not indicated in the Qiagen kit handbook).
23. Aliquot 4 ul of the sample into a new tube for Quality Control (QC) of RNA.
24. Store samples at - 80 C for further use.
Quality Control For RNA
25. Dilute the QC RNA sample 1/50; 2 ul of RNA + 98 ul of NF-water.
26. Place 100 ul of the diluted RNA samples in a disposable UVette and read absorbance using the program for RNA concentration in the spectrophotomer in room 235.
27. Record the dilution factor in the spectrophotomer as per the program to get the right concentration in ng/ul.
28. Read measure OD 260 and OD 260/280 ratio.
29. Run approximately 1 ug of sample (approximately 3 ul) per well on a 1% Agarose gel. Run gel at 100 V for about 40 mins, until the dye front has run approximately half the length of the gel.
30. The gel image must be stored and recorded before submitting the sample for microarray analysis.
31. Bioanalysis of the RNA using Agilent will be done before determining the input RNA concentration for microarrays.
Amplification, Extracted product
|All experiments using protocol P-FPMI-109: (E-FPMI-11, E-FPMI-13, E-FPMI-4, E-FPMI-5, E-FPMI-6, E-FPMI-8)|