Kris, I would say that none are suitable for QPCR except the second one from the left.  I have been working with RNA for over 35 years and the key to good RNA is reducing the time between live tissue and homogenization or snap freezing in liquid nitrogen to a minimum.  Once your tissue is completely homogenized in a guanadinium isothiocyanate based solution, it is safe. Your 28S and 16S bands should be equivalent. Notice how much RNA there is below your 28S and 16S with a large amount at the tRNA level.  These are all signs of RNA degradation and all your QPCR results would be very questionable.  If you are using a kit or doing it the old fashion way (Phenol:ChCl3),the key is still speed.   If you are a beginner at RNA extraction, read about RNase and all the steps necessary to avoid it.  Since you have one acceptable RNA preparation,  I am certain that you can achieve very good quality RNA.

Kris, I would say that none are suitable for QPCR except the second one from the left.  I have been working with RNA for over 35 years and the key to good RNA is reducing the time between live tissue and homogenization or snap freezing in liquid nitrogen to a minimum.  Once your tissue is completely homogenized in a guanadinium isothiocyanate based solution, it is safe. Your 28S and 16S bands should be equivalent. Notice how much RNA there is below your 28S and 16S with a large amount at the tRNA level.  These are all signs of RNA degradation and all your QPCR results would be very questionable.  If you are using a kit or doing it the old fashion way (Phenol:ChCl3),the key is still speed.   If you are a beginner at RNA extraction, read about RNase and all the steps necessary to avoid it.  Since you have one acceptable RNA preparation,  I am certain that you can achieve very good quality RNA.

Hi Kris, I found this picture very useful, I attached also the link.

It seems that your first sample is very degraded, the second is the best.

https://www.thermofisher.com/content/dam/LifeTech/global/life-sciences/DNARNAPurification/Images/1114/f00286.gif

In addition to the above.

If you are suffering from RNA degradation in your samples, you might want to look into the use of materials such as RNA-later etc. These will help to protect your RNA if you can not directly snap-freeze it.

Working fast and clean are key in getting good RNA.

Also note that purity can also be a big issue for RT-PCR analysis. So check with e.g. nanodrop or some other spectrophotometer for A260/280 and 260/230. For clean RNA you want to have a 260/280 of about 2.0 and 260/230 of 2 > . But low concentrations will typically not yield these numbers.

To some extent you can still use degraded samples. But then you really need to use random primers (hexa or decamers) or target-specific primers. 

Thanks for you answers guys. I am aware of the importance of working fast.

I have been following the TRIzol extraction protocol and time between homogenization and addition of TRIzol is 30 seconds. Is that too long? 

My nanodrop readings for all samples were consistently 2.10 (measured 3 times per sample and each reading was between 2.08-2.11). The A260/230 ratio does decrease across measurements though and is within the range of 1.7-2.0

I re-ran the 1st, 2nd and 4th samples (to the right of the empty negative control lane in the first picture) and these were the results. Since I am used to running electrophoresis on wider wells, perhaps I simply used too much RNA for the gel I included in the first post (which had smaller wells). 

Attached are the 1st 2nd and 4th samples. I understand the 1st sample is definitely degraded, so have re-extracted it, but is the 4th definitely also degraded? 

I will re-run the other samples tomorrow in wider wells to see if they look any better. 

I am sorry to disagree with Ruben but low quality RNA is not acceptable for QPCR when following the MIQE rules of QPCR.  Many publications require that any QPCR data be generated using these rules and publishing data that is not valid is useless to the scientific community.   Although, the purity is important, any discrepancies in making the cDNA & running the QPCR will be removed with the reference genes (at least 2). One thing I did not mention in my first response is in reference to the smears that we see from the wells to 26S RNA.  Did you treat your RNA with RNase free-DNAse?  Are those smears due to running the gel too fast?

No, the RNA was not treated with DNAse. 

It may well be due to running the gel too fast or at too high of a voltage?. I used 80V for 60minutes, which produces good images for the larger wells I am used to, but this was my first time running this many samples at once so the wells were smaller. I used less DEPC water and Dye but the same amount of RNA as for the larger wells. 

Kris, I think I see your problem.  You should be homogenizing IN Trizol but what you wrote sound like you homogenize and then add the Trizol.  If you are doing the latter,  then this is the essence of your problem.  As for your RNA preparations, rerunning them will not improve their quality.  We run our RNA for QPCR on a capillary electrophoresis apparatus that analyses the quality of the RNA (Experion by Bio-Rad).  I can definitely say that your samples would not meet the score for QPCR ( 7 of 10 or higher and 9 + for microarray).

You need to treat your RNA with DNase unless you are certain that your primers span a very large intron. If not, some of your fluorescent signal might be generated from amplifying the genomic DNA instead of the cDNA.

I homogenize them for 30s, add Trizol and homogenize for a further 1 minute. 

Should I be adding Trizol immediately and then homogenizing?

In regards to the DNase, the samples are treated with DNase prior to the RT-PCR phase but after I check them on gel.