Hi Mariam!

if you want to check if the kit is working make any kind of measure of the product of it; nanodrop or fluorescence to check the amount of RNA.

Contamination with RNase it´s daily bread so check for contamination sources and clean everyhting.

I think 135v it´s overpowered, I will not pass 130v in any scenario.

hope it helps in way or another!

Hi , I agree with the suggetion above. if you are sure that the extraction follow the protocol, you put always the samples on ice (when needed) and avoid contamination, I will load again your samples, one or two in a small gel tray (after cleaning it very well with NaOH).

In general you run agarose gel 5-6V/cm; I remembered that I run at 50-60V in a small gel tray.

I used a 1% agarose gel.

Also make sure you are using RNAse free water and that the kit components are not expired. If this still does not work you may want to call Qiagen to help you troubleshoot.

Hello @ Mariam,

Wash your cells with PBS and put them in Trizol. I pray that, it will work...

Your RNA samples could be degraded during the electrophoresis.

Mix the related matter in a erlenmeyer (bleach agarose gel) = 1 ml traditional liquid bleach (without parfume), 100 ml 1x TAE, 1 gr molecular grade agarose, 15-20 microliter redsafe, etbr or etc. . Run your gel for 20-25 minutes at the 7 Volt/cm. Analyze your miRNA or total RNA on bleach agarose gel.

This procedure is simple but useful and cheap. You have to use bleach in agarose gel, beacuse, in the presence of low concentrations of bleach, the secondary structure of RNA is denatured and potential contaminating RNases are destroyed. Maybe for this reason (in the absence of bleach) you can't see your RNA samples on normal agarose gel.

By the way, do not run your RNA samples too much time on electrophoresis. Also, warmed TAE or TBE buffer could degrade your RNA  samples easly.

Cheers.

Try to resolve the problem step by step. First, we can't say the RNA was degraded. After the RNAextraction, you should measure the RNA concentration using the nanodrop so that you can confirm whether it is degraded or not. Once you have RNA you can do the electrophoresis and the problem might be in this step, you check the buffers, the reagents.....

Hi Valeria De Arcangelis and Oscar Recasens

My gel length is 27 cm, That's why I choose the volt. to be 135 V.

(5*27)

Isn't that right?

Hi Mariam!

First, you should check RNeasy mini kit is workable. If it is not, please use another new one.

The ubiquitous RNase in nature makes RNA preservation quite difficult, there are some suggestions.

1.As far as possible, a special RNA operation area should be set up in the laboratory, and centrifuges, pipettes, reagents, etc. should be dedicated. ...

2.Always wear disposable rubber gloves during operation. ...

3.Try to use disposable plastic products and avoid sharing utensils such as filter paper, tips, tubes, etc. to prevent cross contamination. ...

4.Ethanol, isopropanol, Tris, etc. used to prepare the solution should use unopened new bottled reagents.

Hi Mariam;

I never used this rule on electrophoresis, because you can do it at 90-110 V, it will take long for the ladder to deplete but the results are quite better.

At higher voltages the buffers can get hot and degrade everything, so i never used the rule Xcm gel * X volts, imagine you have a 30 cm gel, you will burn everyhting down.

Perhaps you have not followed RNase treatment protocol correctly, or there was contamination in work environment might may degraded your RNA sample.

The voltage depends on length between the electrodes.

I recommend that you first should measure the RNA concentration using nanodrop directly after the RNA extraction process.

Then electrophoresis. I usually use 90-110 V or lower for a longer time.

Try to strictly follow the RNase treatment protocol. Hope it works and you have better results.

Hi Mariam Sameh , one easy way to troubleshoot is to start with the gel run. Normally, I clean everything with 1% SDS solution and then wash properly with double distilled water. The gel tray and comb should be cleaned properly. I normally keep a positive control also during an RNA gel; the positive control is usually a sample which I am sure RNA is not degraded before. This will help to understand if any degradation occurred during the gel run. For RNA, I always use 75 V and take a picture after running for 25 min. Hoping this works for you.

RNAse enzymes are everywhere and on everything.

But first, you need to rule out getting a poor yield from your initial extraction. As others said, you need to measure the concentration of your RNA before you run a sample on a gel.

How many cells did you start with? The kits have upper and lower limits for how much you can use in one extraction. Too many cells can be just as bad as too few.

Katie A Burnette

I started with 0.5x10^6 cells

The lower limit is 100 cells and upper limit is 3–4 x 10^6 cells.

Hi Mariam, gel will only show the quality of your RNA extracted, not the quantity and you need both good quality and quantity for further downstream work. First measure your extracted RNA on nanodrop to check its concentration, OD 260/280 and OD 260/230 ratios to get sure about the yield and purity first. For RNA quantification, you can rely on nanodrop readings with good confidence and if you have Qubit assay so is a plus advantage. If they are fine then problem lies in your gel phase, which you need to figure out and if nanodrop readings are bad so revise extraction with caution while taking handsome amount of startup cells. As a rule, a single cell can generate 5-20 pg of total RNA. Remember, high voltage, lengthy runs, heaving loading with samples, all result in gel smearing and smiling in RNA case. Also, on denatured (native) gel, accurate sizing of RNA is not possible because of the RNA secondary structures milder bands often appear above the 28s band and below gDNA bands (if there was no DNAse treatment done). If on native gel you clearly gets only one band either 18s or 28s then it is a go to use that RNA for downstream purpose and if you observe no smearing below the 18s then it is also good to use that RNA. Use always a dedicated gel tank for RNA electrophoresis. You should first clean the tank and its other accessories with 6% bleach or 10% H2O2 or 0.5% SDS or 0.1M NaOH having 1mM EDTA (choose any one of them) then finally wash and rinse with either DEPC-treated water or any RNAse-free water. Only then you can use that tank for RNA work. An ideal RNA gel should run as 5-6 v/cm, 40-65V for 45-50 minutes.

Regards.....

HI Mariam Sameh

All opinions above are important and already give you lots of ideas of where your problem may be ... My advise is about your sample and Joule Heating (If you feel it on your tank buffer)...

One of the major problems with cells for RNA gel electrophoresis may arise from the treatment you have done to the sample or the way you decide to take your cells from the plate or cell culture flask ... I usually do take my cells from the cell culture plate by washing them and already lysing and protecting my RNA direct on the plate. Just after it, I took the solution with lysed cells and nucleic acids from it to an eppendorf microtube.

So, I am telling you that you should evaluate extract/purify your RNA as soon as possible to avoid degradation. It is usually one of the major problems when you find out your RNA degraded.

Secondly, When you end your electrophoresis run, do you feel the chamber heated up? Is the buffer solution hot? If not a bit, your running voltage is ok ... If you feel it hot, use a lower (half you're using now) voltage. Ok?

Best wishes for your experiments.

Hi Mariam Sameh , I am usually very careful when working with RNA (I disinfect the area and pipets, I change my gloves every time I touch outside that area, I use pipet tips with filters that are in a drawer with RNAse-free things). This might seem too much but as Katie said, RNAses are everywhere. Apart from that, I would also make sure that the kit you are using works. You could ask for the kit to someone else or you could try to do the extraction without the kit, which is very easy as well. That way you will figure put if the problem is in your kit. Additionally, I would use a positive control (ask someone for a RNA sample) for the electrophoresis, to make sure this is not your problem.

Hope it helps. Good luck!!

As Marta Lovera suggested try to do RNA extraction with Trizol, chloroform and isopropanol, I prefer doing this method rather than using extraction kit.

I agree with other comments, I usually use Trizol and RNAase zap to clean pipettes and lab bench. It is crucial to work on ice, indeed I also use cool propanolol and sometimes I put the sample with propranolol at -20° for 15-30 minutes, before centrifugation to increase RNA yield. Be sure that DEPC water is not contaminated and keep always RNA samples in the ice when you use them.

Anyway, I think that run conditions are good.

Rosa Gallo @ I agree.

Thanks everybody, I really appreciate your advice and help.

I repeated the RNA extraction but this time I didn't store the cells at -20 but did the extraction immediately after harvesting. And I started with about 2.5 million cells instead of 0.5 million. I got very good bands with good integrity.

So, I think the storage in PBS was the problem and may be the no. of cells was a bit low.

I Run them on 95 Volt for 35 mins

1.2% Agarose