Total RNA isolation with RNeasy MICRO (Qiagen) SPIN Protocol
RNeasy Micro Kit (50) Ref:74004
QIAShredder (250) Ref:79656
NOTE:
High quality of RNA is then eluted in 30μl, or more, of water. P10
Useful for 1(?) up to 0.5×106 cells. Samples maximum 0.5×106 cells using the spin protocol.
For small number of cells homogenisation can be done by vortexing. P23
The binding capacity of the column is 45ug RNA. P39
A minimum number of 100 cells can generally be processed with RNeasy mini columns. P30
All centrifugation must be performed at 20-25oC.
Resuspend dry pellet in 350μl of RLT buffer. Ensure that β2-ME has been added to the RLT buffer less than a month before (add 10ul β2-ME per 1ml of RLT buffer. d=1/100).
Homogenize the samples by pipetting up and down and Vortex several times.
If < 1x105 cells are processed, the cells can be homogenized by vortexing. Make sure no clumps cells are left.
NOTE:
When processing < 5000 cells add 20ng of carrier RNA to the 350μl of RLT buffer. Stock solution frozen at 400μg/ml. Dilute it to 4μg/ml (4ng/μl) and add 5μl to the 350ul of RLT buffer.
Transfer the 350ul lysis solution onto a QIAShredder spin column (purple) placed in a 2ml collection tube. Centrifuge for 2min at MAX speed.
Add 1 Vol (= 350μl) Ehanol 70% to the homogenized lysate and mix well by pipetting.
Transfer the 700μl of the sample into an RNeasy MinElute spin column (pink) placed in a 2ml collection tube. Close the tube and centrifuge for 15sec at 8000g (10000rpm). Discard flow through.
Add 350μl of RW1 buffer into the column. Close the tube and centrifuge for 15sec at 8000g (10000rpm). Discard flow through.
Dilute 10μl of (RNAse-free) DNaseI stock solution to 70μl RDD buffer and mix by pipetting.
Add the 80μl of DNaseI solution to the column and incubate 15min at RT.
Wash with 350μl RW1 buffer into the column. Close the tube and centrifuge for 15sec at 8000g (10000rpm). Discard flow through and collection tube.
Transfer the RNeasy MinElute spin column (pink) into a new 2ml collection tube. Add 500μl RPE buffer into the column.
NOTE:
Ensure that ethanol absolute has been added to the RPE buffer before (add 220ml ethanol absolute per 55ml of RPE buffer).
Close the tube and centrifuge for 15sec at 8000g (10000rpm). Discard flow through.
Add 500μl of Ehanol 80% into the RNeasy MinElute spin column (pink). Close the tube and centrifuge for 2min at 8000g (10000rpm)to dry RNeasy silica gel membrane. Discard flow through and collection tube.
Transfer the RNeasy MinElute spin column (pink) into a new 2ml collection tube. Open the cap of the spin column and centrifugate for 5min at MAX speed.
To elute, transfer RNeasy MinElute spin column (pink) to a new 1.5ml eppendorf tube (cut top and numbered). Add 22μl (can be reduce to 14μl) RNase free water directly to the RNeasy silica gel membrane.
Centrifuge for 1min at MAX speed.
Transfer the 20μl containing RNA in new small eppendorf tubes, which contain 2μl of oligo-dN6 (3mg/ml) –> 2μl/sample. (Promega: Random Primers 20μg, Ref: C118A, £28.98).
Denature 5-10 min @ 70oC.
Shock cool on ice
Prepare the mix for the reverse transcription.
Reverse trancription:
Random primers used : Promega Random Primers 20μg IBR-C1181, 1*20μg, £28.98
Add RT-Mix containing –> 18μl/sample
Per sample 20μl + 2μl + 18μl = 40μl
RNA dN6 RT-Mix
6.375μl H2O
+ 8μl 5X first strand buffer (Promega Ref IBR-M1701)
+ 1.5μl dNTP (10mM) (Invitrogen: dNTP Set 100 mM 4 X 25μmol, Ref IBR10297018, £89.10)
+ 0.625μl RNAse Inhibitor 25 Units (Invitrogen, RNaseOUT™ Recombinant Ribonuclease Inhibitor IBR10777019, 40Units/ul, £62.65)
+ 1.5μl M-MLV 300Units (Promega, M-MLV Reverse Transcriptase, 200 U/ul Ref IBR-M1701)
Mix well using pipette.
1h 42oC (41C, block control and heated lid in Hybaid PCR machine)
Heat 10′ to 90oC to inactivate RT
Dilute to 100μl with TE-buffer if necessary
Store at 4oC (-20oC for longer periods)
Total RNA isolation with RNeasy MICRO (Qiagen) —> PDF