Total RNA isolation with High Pure RNA Isolation kit (Roche)
High Pure RNA Isolation kit (50) Ref: 11 828 665 001
NOTE: Useful for 1000(?) up to 0.5×106 cells. Karine’s advice the Roche kit is much cheaper and way easier to use than the Qiagen kit. However both kits start to have problem below 1000 cells the Roche kit is less Recommended!
Resuspend cells in 200μl PBS. Add 400μl of the Lysis/-Binding Buffer (green cap).
If cells have been frozen in dry pellet add 600μl made of 200μl PBS + 400μl Lysis/-Binding Buffer (green cap). Mix the content well.
Vortex several times and Homogenize the samples by pipetting up and down. Make sure no clumps cells are left.
NOTE: (Facultatif –> When processing < 5000 cells add 20ng of carrier RNA (Qiagen) to the 400μl of PBS/Lysis buffer. Stock solution frozen at 400μg/ml. Dilute it to 4μg/ml (4ng/μl) and add 5μl to the 350μl of RLT buffer.)
To transfer the sample to a High Pure Filter Tube:
• Insert one High Filter Tube in one Collection Tube.
• Pipet entire sample into the upper reservoir of the Filter Tube (max. 700μl).
Close the tube and centrifuge for 15sec at 8000g (10000rpm).
– After centrifugation:
• Remove the Filter Tube from the Collection Tube, discard the flow through liquid, and again combine the Filter Tube and the used Collection Tube.
After re-inserting the Filter Tube:
• In a separate, sterile reaction tube, mix per sample 90ul DNase Incubation Buffer (white cap) + 10μl DNase I. Then add 100μl of this solution to the upper reservoir of the Filter Tube
• Incubate for 15 min at 15 to 25°C
After the DNAse incubation:
• Add 500μl of Wash Buffer I (Black cap DO NOT FORGET TO ADD ETHANOL 20ML!!) to the upper reservoir of the Filter Tube assembly and centrifuge for 15sec at 8000g (10000rpm).
• Discard flow through and combine Filter Tube with the used Collection Tube
After the first wash:
• Add 500μl of Wash Buffer II (blue cap DO NOT FORGET TO ADD ETHANOL 40ML!!!) to the upper reservoir of the Filter Tube assembly and centrifuge for 15sec at 8000g (10000rpm).
• Discard flow through and combine Filter Tube with the used Collection Tube
• Add 200μl of Wash Buffer II (blue cap DO NOT FORGET TO ADD ETHANOL 40ML!!!) to the upper reservoir of the Filter Tube assembly and centrifuge for 2min at MAX speed (13000rpm) to remove any residual Wash Buffer
Discard the Collection Tube and insert the Filter Tube into a clean, sterile 1.5ml microcentrifuge tube.
To elute the RNA:
• Add 50–100μl Elution Buffer (transparent cap) to the upper reservoir of the Filter Tube
• Centrifuge the tube assembly for 1min at 8000g (10000rpm).
• When starting from very small numbers of cells only use 40ul Elution Buffer to the upper reservoir of the Filter Tube and clean the column twice with it. So put it back to the column and centrifuge again the tube assembly for 1min at 8000g (10000rpm).
–> The microcentrifuge tube now contains the eluted RNA. Either use the eluted RNA directly in RT-PCR or store the eluted RNA at –80°C for later analysis
Transfer the 40μl containing RNA in new small eppendorf tubes, which contain 4μl of oligo-dN6 (1mg/ml) –> 4μl/sample. (Promega: Random Primers 20ug, Ref: C118A, £28.98).
Denature 5-10 min @ 70oC. –> Shock cool on ice
Reverse trancription:
Random primers used : Promega Random Primers 20ug IBR-C1181, 1*20ug, £28.98
Add RT-Mix containing –> 36μl/sample
Per sample 40μl + 4μl + 36μl = 80μl
RNA dN6 RT-Mix
12.75μl H2O
+ 16μl 5X first strand buffer (Promega Ref IBR-M1701)
+ 3μl dNTP (10mM) (Invitrogen: dNTP Set 100 mM 4 X 25umol, Ref 10297018, £89)
+ 1.250μl RNAse Inhibitor 25 Units (Invitrogen, RNaseOUT™ Recombinant Ribonuclease Inhibitor IBR10777019, 40Units/ul, £62)
+ 3μl M-MLV 300Units (Promega, M-MLV Reverse Transcriptase, 200 U/ul Ref M1701)
Mix well using pipette
1h 42C (41C, block control and heated lid in Hybaid PCR machine)
Heat 10′ to 90C to inactivate RT
Dilute to 100μl with TE-buffer if necessary
Store at 4C (-20C for longer periods)
Note : Prog 5 RI