Absorbance values will only indicate that you have got nucleic acids in your prep, but they won't tell you anything about the size of the species or their identity . It sounds like your prep does not contain RNA or that it is heavily degraded. That you don't even see 18 and 28S RNAs is surprising, because, owing to their secondary structure, these are usually more resistant to RNases than mRNA. Thus, it is not surprising that you can't get cDNA. I would suggest that you take great precautions to avoid RNase contamination : work with autoclaved solutions, preferably treated with diethyl pyrocarbonate, use disposable plasticware or baked (180 °C) glassware and wear gloves throughout the isolation procedure.  

Probably as it has been said before your RNA is heavily degraded.Other option is that your RNA fragment has gone away if its small an you put it on electrophoresis gel too long.When I work with RNA I take the cosiderations said before and try to use RNA materials only for this purpose and use NEW electrophoresis buffer made at the moment.

Good luck.

Nanodrop 260/280 and 260/230 reading shows absorbance of nucleotide. As in gel image clearly you can see that RNA is degraded and if there also concentration is very low. So I will suggest to perform new total RNA isolation and this time after homogenization just sure that Trizol should not change. If changing to white add little more Trizol and try to maintain pink color. And during re-suspension step try to use new autoclaved DEPC water( Or I can suggest to re-suspend RNA pellet in tris-EDTA)

Your RNA could be degraded on the gel, but having no cDNA indicates that it  might not be the case.

For good RNA extraction from the tissue, the most important is what yo do to the tissue BEFORE  adding Trizol and how well and quick you homogenize your sample in Trizol. Degradation occurs before the action of strong RNAse inhibitor present in Trizol (4M guanidine isothiocyanate). Check the the steps before Trizol in your protocol. And before running RNA on the agarose gel, soak the gel apparatus, the comb and gel bed in 1% SDS solution for 1h, to inhibit RNAses. After that, rinse with ddH2O and run regular TAE gel. Do not forget that RNA needs to be denatured for 5 min just before loading, in an SDS-containing loading buffer, otherwise it will get degraded.

Good luck!

Evguenia

what is the ladder you are using, and what species of RNA are you working with. If it is from flies or some bacterial species like D.radiodurans, based on the degree of separation of your ladder, you will see one band at around 2KB ( fly 28S is split into 2 bands roughly the size of 18S each)  unless you run it long enough you wont see 3 bands), but if it is from others, like humans, i would definitely repeat the prep, and make sure there is no RNASe contaminations. BTW you cannot see cDNA on a gel. 

In addition to the comments concerning RNA degradation-I can't tell if any of the lanes have a sizing ladder. Also the dye you're using (looks like bromophenol blue) could mask the bands. I changed to a dye containing Orange G and xylene cyanol (Fermentas/ThermoFisher) to help with this problem.  Change dyes, add a sizing ladder in the relevant size range, and run gel longer.

Please go into detail of how you get the tissue, how it is stored prior to isolation, and specific on tissue disruption in the Trizol. That might help with answers you receive. I'd like to offer advice but need a little more background.

I isolated the RNA from bull edpididymis. The tissue was stored in RNA later solution till isolated. Yes, the first lane is DNA ladder, 2nd lane is a control GAPDH RNA, and the next 3 lanes are isolated RNA loaded with BPB (app.1.5ug)

Thank you @Vipul Batra for the additional info. Based on what I see the gapDH RNA is a nice distinct band as expected so the get is denaturing either glyoxal or formaldehyde. Leads me to ask about the tissue isolation. How soon was the epididymal tissue placed in the RNA later? Which mechanical mechanism was used for tissue disruption, homogenizer or bead beater, etc.? You mention the usual 18 & 28S bands; have you previously used this RNA method on this tissue type with success? Apologies for all the questions, just trying to narrow down the possible causes.

Within 30 min, I suppose. that includes time spent on washing with alcohol, PBS, and then dissecting and washing again before pouring in RNA later solution. 

P.S. the gel is not denaturing. DEPC, TAE.

I got very faint but sharp bands when I ran a PCR of the undiluted DNA which I made from this RNA, with GAPDH primers . Also, qPCR with GAPDH primers yielded Ct at 22.80 which is good. However, any other primer didn't work. Housekeeping gene's expression  is always more may be that's why ....

Sure I will, Thank you.

Another suggestion is to thinking about the reagents you are using during your dissection.  Use ethanol that is used only for RNA work.  If you are diluting down from pure ethanol, do the dilution with RNA-free water.  Your PBS should also be made RNAse-free, and only be used for RNA work. 

Make sure that your dissection area is prepped for RNA work, your scalpel, forceps, and other materials should all be cleaned with water, RNase-free ethanol, and then RNase-Away (or equivalent) before you start dissecting, and between samples.

Good luck!

Hi Vipul,

Many good suggestions on RNA prep and extraction procedures already so I will leave that part.

Gel is a good quick quality control step but it's not sufficient for telling you the quantity and the quality. I highly recommend taking a 2ul aliqot of your final extract and sending it to Bioanalyzer - Many time I have had excellent Nanodrop results but the RNA was utterly degraded, as the Bioanalyzer showed.

Good luck!

This is normal RNA and does not appear to be degraded.

@Jai: Is it normal? How can you say so ?The cDNA didn't yield any results. 

If cDNA didi not yield any result then it is the fault of the DNA polymerase

I gave the tissue sample to another lab for RNA isolation. They got the same kind of RNA when it was ran in gel. So, now I think the problem has been identified that the tissue was problematic. I procured the sample within 15-20 min of removal from an abattoir. Washed it in absolute alcohol, then with 1xPBS prepared in DEPC water. Then the epididymis' parts, V.D. & rete testis  were removed within 15 min and the samples were once again washed in alcohol and pbs before being immeresed in RNA later solution. It was transported with ice packs in 2 hours and then stored at -80 deg.