Referring to the attached image, has the RNA extracted from the FFPE sample been degraded? The lanes of agarose gel are as followed:
1) Lane 0: 50bp ladder
2) Lane 1: negative control (-RT)
3) Lane 2: PCR product (1:1 dilution ratio)
4) Lane 3: PCR product (1:5 dilution ratio)
5) Lane 4: PCR product (1:10 dilution ratio)
6) Lane 5: Positive control
FYI, we are detecting beta-actin (housekeeping gene) from the FFPE sample. Based on the result obtained from the agarose gel, there is no beta-actin band being detected as its product size claimed. However, a beta-actin band was detected as the upper band for positive control, while the lower band detected should indicate that the RNA extracted has been degraded. In fact, both diluted sample and positive control did show the second lower band. However, we did not check the RNA integrity using 28s and 18s. Based on the agarose gel itself, what do you think?
Thanks
Seng
Hi,
I did not know whether the tissue is properly because it is delivered directly from hospital. Yes, I extract the total RNA using a Qiagen commercial kit called RNeasy FFPE kit (Cat no.: 73504). FYI, the RNA purity is 1.863 for A260/280 and 1.889 for A260/A230.
RK, did u meant the main cause of could not get the band of beta-actin is the RNA extract is degraded and the quality is not good?
How long is your amplicon. You can be sure that your RNA from the FFPE will be degraded to a large extent and the success of your amplification is as is pointed out above the way that the sample is handled both before and during FFPE procedure. Furthermore the length of your amplicon is really important, as you cannot expect to have many long RNA sequences. The shorter amplicon the better.
Adduction, loss of poly A tails, methylol bridging (due to HCHO action), and poly [dT]n priming rather than "degradation" per se could likely be the problem here...
If you primed your PCR with poly [dT]n, it would likely fail and thus no product - due to loss of poly A tails on the b-actin transcripts. Thus, if the lower single bands in lanes 3,4,5 are small enough - they may indeed just be primer dimer and not b-actin product (as stated above). Try using random hexamers or random nonamers instead of poly [dT]n priming. They will prime the body of any extant transcripts and won't assume that poly A tails are still there... available for poly[dT]n priming...
if you have sufficient RNA, load it on denaturing agarose gel and analyses its integrity.
if the whole lane below 5-6 kb show florescence eans your RNA is not degraded. and if it is producing florescence around or below 500 bp, the RNA ay be degraded of contains small fragments (due to partial degradation) only..
if you have sufficient RNA, load it on denaturing agarose gel and analyse its integrity.
if the whole lane below 5-6 kb show florescence means your RNA is not degraded. and if it is producing florescence around or below 500 bp, then RNA may be degraded or contains small fragments (due to partial degradation) only.
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