siRNA detection - i'm totally lost (Jan/31/2005 )

I'm trying to detect sirna transcribed in cellulo.
I use an H1 promoter as described in the litterature to make transcription of a siRNA. It seems efficent due to the fact the targeted protein is now at an undetectable level on a total cellular proteins extract. I want now to detect the relative quantity of the siRNA that are in cell.

I have purified total RNA by using TRIZOL from invitrogen, and concentration of extracts are between 2.52 and 7 µg/µl, depend on extract. I separated rna using a denaturing 15% polyacrylamide / 7M urea gel, and transfered RNA on hybond XL (amersham) membrane.
Then i colored the membrane using ethydium bromide and this shows an efficient trasfert.

For the detection of siRNA, i want to make an 5' end labeled probe, by using T4 polynucleotide kinase and P32-alpha-dATP.
I'm labelling about 500ng of probe and purify the probe, using nucleotide removal kit from quiagen. After purification i run 1/30 of the purified labeled probe on 15% polyacrylamide / 7M urea gel and exposed the gel to a biomax mr film from kodak. The probe is labeled.

I hybridize membrane and probe overnight in a apropriate oven at 37°c (when the tm of my labeled oligo is at 60°)

I'm not able to detect mi siRNA, and not able to detect the U6 snRNA (my loading control).

I've tried to reduce time-lenght of the differnt washes, reduce the temperature of the washes. The only result of that is more noise on the mmebrane.

Can please anyone help me?

I would like to tell you that my lab can't buy any detection kit.
Many thanks

I'm not sure what your washing conditions are. It could be that you're washing your probe off the membrane. When I used to routinely probe for U6 RNA I used to hybridise is SES1 buffer (0.5 M NaPO4, 7% SDS, 1 mM EDTA) at 37 degrees. Membranes were then washed twice for 5 min in 6x SSPE buffer (20x SSPE buffer is 3.6 M NaCl, 0.2 M NaH2PO4, 0.02 M EDTA pH 7.4) followed by 2 washes for 20 minutes in 6x SSPE pre-warmed to 42 degrees.

In addition, to clear up your probe following labelling, no need ot buy or use a nuceotide removal kit - just pass it through a 2 micron filter

Let me know how this compares to your current wash protocol and I'll see if I can help further.

Here is a more detailed protocol

I have purified total RNA by using TRIZOL from invitrogen, and concentration of extracts are between 2.52 and 7 µg/µl, depend on extract.
I separated rna using a denaturing 15% polyacrylamide / 7M urea gel, and transfered RNA on hybond XL (amersham) membrane.
Then i colored the membrane using ethydium bromide and this shows an efficient trasfert.

For the detection of siRNA :
Total volume : 30µL
10x T4 Polynucleotide kinase (T4PNK) buffer 3µl
500ng of probe (concentration : 578ng/µl so i use 0,864µl : is it too concentrate to get a good quantity?)
25 units of T4PNK
2µl gamma32P-ATP 6000Ci/mmol
Probe and the appropriate volume of buffer + water are denaturated 10' at 55°C . then ATP and enzyme are added band all the mixture is let at 37°C for one hour.

The Membrane is preincubated at 37°C in :
formamide 50 % (10ml)
SSPE 5X (5ml SSPE 20X)
Denhardt's 5x (2ml Denhardt's 50x)
SDS 0.5x (1ml SDS 10%)
ssDNA 500µg (50µl at 10 µg/µl)
for minimum 2 hours (usually 2h30).

Pre hybridization solution is chaged for hybridization solution :
same composition exect there is no ssDNA and the purified probe is added
I've measured the radioactivity of 1/30 of the solution of the purified probe and get 770cpm for U6 probe and 450cpm for my specific probe.

I hybridize overnight in a rolling oven (i don't know the exact word in english)

Washes :
First protocol : i used last year :
2X SSC / 0.05% SDS : 2 washes at room temperature, 20' each
0.1X SSC / 0,1% SDS : 2 washes 20' RT

As i didn't observed any result i changed for this one :
1X SSC / 0.05% SDS : 2 washes at room temperature, 10' each
0.1X SSC / 0,1% SDS : 2 washes at RT, first one 15' and second one : 20' RT


I'm not able to detect mi siRNA, and not able to detect the U6 snRNA (my loading control). but i detec unspecific (background or as i call it "bad noise"

could you tell me is the ses 1 buffer is less restrictive than my conditions? For the further washes, you use 6X SSPE, instead of me (1XSSC/0.05%SDS). I was wondering is your solution less restrictive as mine? Is t possible that sds washes out my probe but not the non-specific radioactivity?

I'm totally lost as i said in the forum so anything could help.

Many thanks.

i don't know the interest of wash at 42° as you said your hybridization is at 37°...

I have two more questions :

first what is the pH of the hybridization solution

second : what is the temperature of your washes ? nothing mean room temperature ?

anything could help.

for the pH of the phosphate bufer see

http://www.msu.edu/user/eisthen/lab/method...recipes/PB.html

do you make a pre hybridization?

Hey, how did you transfer RNA from PAGE to HYbond-XL? is Hybond XL good for siRNA transfer?

hi
thanks for your reply!

i transfert siRNA to hybond xl membrane by an electrotransfert, in constant amperage 3mA/cm² during 45' to 1 hour. But i don't have informations about the suitability of hybond XL specifically for siRNA. However, it seems being used in litterature.

What do you think in particular?