detect siRNA derived from shRNA construct - (Sep/26/2007 )
Is there anyone has the experience in detection of siRNA of shRNA expression construct via northern hybridization?
I have failed in such detection, and looking for advices for it...
at first you need to check with a U6 promoter or H1one which serves at reference.
second : migration and transfert in 1X TBE
transfert can be done at 5V overnight 4°
then uv cross link should be nice
baking 2h 80°
do you use a hybond N+ membrane ? or equivalent ? hybond xl membranes are not suitable for this
quality RNA : may you post a picture of your gel colored with EtBr before transfert ? did you check quality of your extraction protocol with agilent technology (it has a rank note which is nice to evaluate ; with cells, 9.5/10 is a minimum, also a colleague using trizol get 10 regulary)
probe labelling : for increasing labeled proportion of the probe, label 10pmol of oligoniucleotide by T4PNK using gamma atp 6000Ci/mmol
finally, you can artificially enrich the small RNA proportion inan easy way :
using a column kit, apply it on a column. it retains only >100bp RNA !
Use the flow through :
add 1volume of phenol chloroform at pH 4.7 homogenizes and keep on ice 10'
separate phases by spinning (8000g 4° 10')
then pipett upper phase, add glycogen (1µl of 100mg/ml) as carrier
add 1volume isopropanol
homogenize and let sit on ice 5'
spin 10000g 4°
wash with 70° ethanol
resuspend in nuclease free water.
Thanks for your help; you did input valuable useful information
Actually, I need the validation of siRNA derived from our new shRNA system. I have the experience in Northern hybridization of miRNA, however, since I failed in detection of expected siRNA derived from our shRNA system, therefore, I wonder anyone have experience in detection of siRNA for shRNA construct. I’d feel the detection of siRNA derived from shRNA construct is different from miRNA.
what is the promotor you use for your sRNA?
did you try miRVANA ? it's a noce one.
also a test in which artificial enrichment is done should be good
It is not commonly used plIII promoter, and seems work based on the analysis of siRNA’s target gene, while, not sure the existence of siRNAs
You are right, I might enrich small RNAs for blotting…
well i mean you should do experiments using also a pol III promoter to be sure that you can detec siRNAs derived from shRNAs from your technique. If the answer is no, you have a pb with your manip.
If the answer is yes, then it's because the quantity of siRNAs is too low.