You obviously have some form of contamination. Aside from cultured cells, there's very little in cell-culture wells after washing with PBS that should contaminate your samples. Look into your technique, and eliminate any potential sources of contamination.

A 24-well plate is good enough to yield high amounts of RNA. I regularly extract RNA from 24-well plate and get very high yield of up to 1000 ng/uL per well when my HEPG2 cells are confluent, and high purity using HiYield RNA Extraction Kit from RBC Bioscience. Any good RNA isolation kit should also give you similar results if you follow the protocol carefully, and avoid any potential sources of contamination from your surrounding.

What is recommended when you extract RNA from a culture plate is that you add the lysis buffer directly onto the cells in the wells, and proceed as instructed by your protocol. Otherwise, you can wash the cells on the plate with PBS, cover the plate and seal using parafilm, then store at -80 degrees (i have stored my cells this way for over 6 months). This way you dont lose cells (and RNA) during scraping or trypsinization of the cells from the plate, and chances of contamination will be slim.

Make sure to check if you are required to add 2-Mercaptoethanol to your lysis buffer. Most column based RNA extraction kits will also recommend that you spin the column for 3-5 mins after discarding the flow-through from your last wash step, before eluting your RNA. This removes any left-over reagents on the column. Finally, a common mistake when using column-based extraction kits is that RNA is mistakenly eluted into the collection tubes that was used to collect and discard the waste reagents during extraction; dont forget to use a new sterile RNAse-free collection tube to elute your RNA.

Hope this helps!

why cant you use trizol for RNA isolation. It would be better if you use 100 mm or 60mm dishes where you can scrap easily / cell number will be more incase of flask it would take more time. hope this will work out for you

Thanks Utkarsh, I have heard of Trizol, but i have never tried it. I just need to understand where I might be missing the point with my extraction. Thanks

Small amount of material is always a challenge when isolationg RNA.

Are you able to use 12-well plate or 6-well plate for your experiments?

Some companies offer kits for RNA isolation from small amount of material (the difference is e.g. the size of column for elution). Another idea is to use a carrier molecule for RNA when performing the isolation.

forgot EtOH?

I think that two wells of a 24 well plate is too small for RNA extraction. In addition, the elution of the column need an important volume of water or TE and dilute your RNA. You could try trizol, because the last step is a precipitation and not an elution. You could add some glycogene as a carrier during precipitation steps.

try for Trizol method of extraction,it will surely help u out

What do you use for elution... Double distilled water has a pH of 4 which interferes with 260/280 measurement.. I would also go for Trizol or PCI extraction...

You obviously have some form of contamination. Aside from cultured cells, there's very little in cell-culture wells after washing with PBS that should contaminate your samples. Look into your technique, and eliminate any potential sources of contamination.

A 24-well plate is good enough to yield high amounts of RNA. I regularly extract RNA from 24-well plate and get very high yield of up to 1000 ng/uL per well when my HEPG2 cells are confluent, and high purity using HiYield RNA Extraction Kit from RBC Bioscience. Any good RNA isolation kit should also give you similar results if you follow the protocol carefully, and avoid any potential sources of contamination from your surrounding.

What is recommended when you extract RNA from a culture plate is that you add the lysis buffer directly onto the cells in the wells, and proceed as instructed by your protocol. Otherwise, you can wash the cells on the plate with PBS, cover the plate and seal using parafilm, then store at -80 degrees (i have stored my cells this way for over 6 months). This way you dont lose cells (and RNA) during scraping or trypsinization of the cells from the plate, and chances of contamination will be slim.

Make sure to check if you are required to add 2-Mercaptoethanol to your lysis buffer. Most column based RNA extraction kits will also recommend that you spin the column for 3-5 mins after discarding the flow-through from your last wash step, before eluting your RNA. This removes any left-over reagents on the column. Finally, a common mistake when using column-based extraction kits is that RNA is mistakenly eluted into the collection tubes that was used to collect and discard the waste reagents during extraction; dont forget to use a new sterile RNAse-free collection tube to elute your RNA.

Hope this helps!

I would look at the simple fixes with the kit first. Daniel Guendel is on the right track with the Ethanol. Another common problem is not lysing the cells properly. I usually use Qiagen kits for RNA if I'm wanting mRNA and not miRNA (in which case Trizol is the best way to go), but I usually double or triple the lysis time (on ice, of course) and have seen that increase the yields significantly.

Qiagen RNeasy kits work well for isolation of total RNA from 24-well plates. We do this routinely for primary cells and, depending on cell density, recover >300 ng/uL RNA which is more than sufficient for quantitative RT-PCR. As one or two others have alluded, insufficient lysis is likely part of the problem. After adding RLT buffer to the well, be sure to completely dislodge the cells from the bottom of the well (and importantly from around the edges) by scraping thoroughly with a pipette tip. Pass the RLT lysate through a sterile 23G needle 3 times to break the cells open before adding ethanol and doing column puriifcation. Finally when eluting the purified RNA from the column use smaller elution volumes (25-30 uL is fine) and leave the buffer on the column for 5 mins before centrifuging. Good luck!

Thanks everyone.Daniel, I did not forget EtOH. I'd like to continue with the Promega kit, I got a good yield from using a culture flask. Others have gotten good yields using 24 well plates, but I'd like to use 12 well plates as suggested by Agnieska and Stephane. I wanna improve on cell lysis (Raymar) how do i do that? I use lysis buffer and B-mercaptoethanol. Can I repeatedly vortex and place on ice for few mins? Mustapha, I vortex to lyse cells, so cant apply lysis buffer directly yo the wells. I wash with wash buffer at the end. I will try leaving the buffer at culumn for 5mins before centrifuging. Norbert, i use RNase free water for elution.

I don't know what is your end point but I can assure you that you will get enough RNA from single 24 well plate. You should have 100-200ng/ul and which is more than enough for cDNA synthesis and qPCR. You can go with quiagen mini kit with triazlole followed by lysis with either tissue lyser or syringe lysis. Then appropriately follow kit protocol.

Good luck

Do you use the cells for anything other than the RNA extraction? If not, my point is, you can directly extract the RNA from the culture plates thereby reducing chances of any contamination in the process.

Yes, I second the opinions above- Lysis buffer directly into the plate ! Ideally trizol, incubate for 2 min on ice, scrape, remove into tubes including 10x shear mixing with the pipette, incubate for another 5 min at room temp then go to the kit. We use 750ul Trizol for a 6 well plate so I suggest scaling down to no less than 250 (you loose to much in the transfers otherwise). Vortexing is not a suitable method for lysing as the eddies in the fluid don't generate the right kind of forces. You may have gotten a good yield from the culture flask due to the shear numbers of cells that you had, probably millions. Working with 24 well plates you are going to be (at most) in 100's of thousands so you need to squeeze all the RNA you can out of each cell. Good luck !!

Thanks a million Bobbi !

A little late but anyway. Keep in mind that RNA and DNA adheres to the surface of certain kinds of plastic. So I recomend to lyse your cells in low binding tubs and not within your culture flask or culture plate. Thats important especially if your worke with low cell numbers. To transfer your cells form your culture flask to your tube wash them twice with ice cold PBS and than use some of the PBS and just scrape your cells form the surface, centrifuge and thats it. Good luck.

Thanks Henri!

Hi Phil,

I am using the Qiagen Rneasy kit, below is my customized protocol, I do get high yield of RNA and the A260/A280 and A260/A230 are met the desired values.

• Add 350µl RLT, use 25G needle to resuspend (X10-20 times), and leave at RT for 5 minutes

• Add 350µl 70% Ethanol, and vortex to mix well, quick centrifuge after vortex

• Transfer the mixture to column, and spin at 10,000 rpm for 15 seconds

• Add 700µl RW1 , wash around the column, then spin at 10,000rpm for 15 seconds

• Add 500µl RPE, wash around the column, then spin at 10,000rpm for 15 seconds

• Add 500µl RPE, wash around the column, then spin at 10,000rpm for 2 minutes

• Transfer the column to new collection tube, heat with heater at 55°C for 15minutes.

• After that , spin the tubes at full speed for 5 minutes

• Transfer the column to 1.5ml collection tube

• Add 40µl heated Rnase water to column (55°C), and incubate at heater for 5 minutes (55°C)

Elute the RNA by spin the column by spinning the column at 10,000rpm for 1minutes

As Henri`s suggestion,  try scrape the wells with cell scraper or bended 20ul pipette tips after lysis the cells.Good luck.

Ks Lim Hi, KeeSiang

I'm reading your comment that is very helpful!

However, in the step"Transfer the column to new collection tube, heat with heater at 55°C for 15minutes". Wouldn't this cause the degradation of your RNA. I have never seen a protocol to heat your RNA during the RNA extraction.

Could you clarify it please?

Thank you very much!

Best regards,

Phraew Sakon

The purpose is to make ethanol evaporates faster. You can use vacuum centrifugation if you have. For me, this works all the time. I manage to use the heated RNA to make cDNA. qPCR results look good too.