Hi Juan,

did you check the RNA quantity and quality (absolutely important!) using the Qubit and/or Bioanalyzer? From my experience, only RNAs with comparable RINs as provided by the Bioanalyzer yield comparable results and since the quantification of the Bioanalyzer is not too good, I would use the Qubit for proper quantification! The Nanodrop will measure all nucleic acids including fragments or nucleotides whereas the Qubit uses a ssRNA-specific dye. We do that for qPCRs and are very successful!

You may check the amount of total RNA in the gel by staining the agarose gel with etidium bromide before the transfer to the membrane. You should have the same intensity in all lanes if you have loaded the same amount of total RNA. It can also be a problem of transfer to the membrane...

I also recommend staining of your total RNA samples in a gel with ethidium bromide or other nucleic acid staining dye. You can even blot the same gel afterwards. There are commercially available methods, which separate your RNA samples and directly calculate your sample from the stain of rRNAs (see URL http://rna-integrity.gene-quantification.info/).

Hi Juan,

did you check the RNA quantity and quality (absolutely important!) using the Qubit and/or Bioanalyzer? From my experience, only RNAs with comparable RINs as provided by the Bioanalyzer yield comparable results and since the quantification of the Bioanalyzer is not too good, I would use the Qubit for proper quantification! The Nanodrop will measure all nucleic acids including fragments or nucleotides whereas the Qubit uses a ssRNA-specific dye. We do that for qPCRs and are very successful!

I think the better option which i usually is to quantify your RNA samples first with nanodrop. Then add one ug of RNA sample in agrose gel and then do ethidium staining. Evaluate the bands according to their intensity.

I agree with the other responders. Make sure that the samples are loaded evenly prior to transfer, you can add a small amount of ethidium bromide to your sample buffer prior to adding it to the samples. I used to precipitate my RNA prior to adding sample loading buffer so that all volumes would be the same when it came time to load. Do you expect the 16S to be smeary, gel buffer may be a little off also and this makes it very difficult to quantify relative expression if the bands are not sharp. Also make sure your electrode wires are straight, the bands going across look a little wavy. Good luck.

You must quantize well the RNA on an agarose gel before performing the Northern-blot. You must load 100 ng and 200 ng of RNA per sample on an agarose gel stained with ethidium bromide and see if the quantization using the NanoDrop is correct. then you have to load 12 micrograms of RNA (quantized correctly) on denaturing agarose gel (formaldehyde - formamide) stained with ethidium bromide to see if indeed we loaded equal amounts of RNA.

I also suggest to do a more in dept analyses of the RNA quality by Qubit or bioanalyzer before running the samples. An alternative method could be to use the same amount of cells for each lane. If the total amount of RNA is not constant over time due to the treatment (or in the comparison between treated and untreated samples) this might also influence the relative amount of your specific mRNA.

I would nanodrop the samples and then adjust concentrations so that they are all supposedly equal and then check them for intensity on an agarose gel with formamide in loading buffer to prevent degredation. And as pointed out above use freshly autoclaved buffers.

I think variation while loading the samples is something unavoidable. You can rather try to normalize your results to the control band than to try to have the same intensity in all wells.

I agree with everyone that it is necessary to quantify RNA concentration by nanodrop, Qubit, or gel stain. But the RNA isolation method is also very important. The recovery of some methods are not very consistent, especially for small RNA during the ethanol precipitation. So, to avoid the ethanol precipitation, you might consider to use column-zol method, which combines TRIzol with column to bypass ethanol precipitation. Also, if you have problem getting enough cells after drug treatment, you might consider to use RNA Tini Spin column. It is designed to isolate or concentrate RNA from small amount of cells. Both RNA mini and Tini columns are sold in bulk and you can use them with TRIzol or Qiazol to save money. Please let me know if you need protocol or have any questions.

I agree with the suggestions of others. Please kindly also check the agarose gel preparation steps. Some times all the wells may not be at the same level due to comb level. This generally happens when the surface where the casting is made is not proper or the casting is not done properly. Diluting all the samples to the same concentration also allows to reduces the errors. The volume of sample loaded must be made to near equal volumes as more concentrated samples tend to settle down in the well bottom early than very diluted samples. Please don't leave the samples long in the gel while running. Note the gel die when it's running and after the desired bands appear (or check Gel front), take it out for analysis from the buffer as immediately as possible. This helps in reducing diffusion of the samples in the gel (crescent moon shaped bands appear) as well as in the tank buffer.

You should quantified it before load the gel. I used do it by spectrophotometer and load the same. You can also mixed it with BrEt before (the same volume in all samples) load and see the RNAr to stimate the load

Normally, you could measure the optical density at 260 nm and calculate the amount of RNA. To see the quality, you measure the relation 260/280 nm of each sample. Otherwise, you do ethidiumbromide staining of you gel to see whether the bands are equally stained..

The wavy looking bands might be also due to salty or undissolved samples. For Northern analysis the best extraction method would be cesium chloride gradient. Also magnetic beads or columns would be OK. Prior to transfer the gel is transiently stained to check for the integrity of RNA via looking at 28 and 18S rRNA in case you use the total RNA. Using internal and external controls as appropriate from the extraction step till the quantification step is mandatory to locate the problem.