What is the recommended RNA extraction protocol from cultured adherent human cell lines?

12 December 2014 16 1K Report

I'm planning for culturing SCC cell line & want to know the best technique to extract RNA from them & recommended protocol or extraction kit prior proceeding qPCR steps.

I'm still confused among numerous commercially-available kits, some can work directly on the cultured cells without lysis/homogenization steps till yielding cDNA, others require lysis of adherent cells with/without homogenization....

which is better & easy in manipulation since it is the first time to perform a PCR assay.

Thanks

Guillaume Vidal

Hi,

TRIzol reagent is easy to use and works well.

Gianni Gori Savellini

I generally use total RNA extraction kit from macherey-nagel. It's simple, you can directly lyse adherent cells on the dish and then move to epps and columns. However keep in mind to always treat eluted RNA with DNaseI!!!

Ajay Kumar

Trizol works very well with cultured adherent cells if you have a good cell amount. Take care to follow proper temperature range in that and use of chilled reagents whereever required. In case you have very low no. of cells you can go for Qiagen RNA isolation kit. That works very well. But if u have enough cell no. don't opt for costly kits. Trizol extraction is best..Hopeit helps..!

Basant Hamdy

Thanks all for your feedback.

Marc-David Ruepp

It depends a bit what you would like to afterwards. Trizol works normally very nice but the RNA fraction can contain plasmids that you transfected before or slight gDNA contamination. If your qPCR assay spans exon-exon junctions this is normally not a problem (include minus RT controls anyway!). However if your qPCR assays do not span exon-exon junctions you have to perform DNAse treatment afterwards. In this case using a column based isolation method might be nicer, as there you can do on-column DNAse treatment and you do have to do the second purification to get rid of the DNAse.

Achilleas Mitrakas

Trizol works very well but rna isolation kit (Macherey-Nagel) is easier and faster!

Bram Piersma

Hi Basant,

I agree with the previous answers in that Trizol is quick and easy but may result in a contamination with DNA. We used to buy the Qiagen kits but they are quite expensive, so now we switched to a cheaper kit from Favorgen (a company from Taiwan). These kits do work very well and give the same yield, quality and purity as Qiagen kits, for about 40% of the price.

Danijela Markovic

I tried many different kits that are easy to use, but if you want really good RNA quality and yield from not so many cells I would recommend mirVana™ miRNA Isolation Kit from Ambion. It is bit smelly since you need to use phenol and it takes bit more time than quick spin kits. If you have lots of cells you could use a kit from Zymo (I think they are the cheapest). In any case DNAse treatment is a must. Hope this helps.

Basant Hamdy

All suggestions are great. Thanks all

Basant Hamdy

Dear colleagues,

i'd like to know your opinion in the attached kit handbooks... which one is better for yielding good quality RNA for conventional qPCR analysis?

Rania Darwish

Well this depends on your next experiments, whether your goal is to extract total RNA or you would like to have in your extract small non-coding RNAs. From my experience, go for Trizol. It is a very easy protocol, inexpensive, and takes a little time.

Good Luck.

Basant Hamdy

Thanks everyone

wish me good luck

Elena Stavenschi

TriReagent from sigma works extremely well also.

Basant Hamdy

Thanks Elena

Dhafer Al-Koofee

There are many methods and kits used for extraction involving TRIZOL reagent, but may a problem with not completely dissolved of extracted RNA, and advantage give high yield reach 200 micrograme at the end of protocol

Sadegh Zarei

RNA isolation procedure for cells

Using at least 106 cells, aspirate the media and wash once with ice cold PBS (1–2 ml).

Aspirate the PBS (remove as much as possible) and add 1 ml TRIzol.

Scrape the plate briefly, then remove the TRIzol with a pipette and deposit the TRIzol/cell lysate into a 1.5 ml Eppendorf tube.

Leave at room temperature for 5 min.

Add 250 µl chloroform and shake the tube vigorously for about 15 sec.

Leave at room temperature for 5 min.

Centrifuge at 10,000 rpm for 5 min.

At this point, there will be three layers in each tube: Top layer: clear, aqueous Middle layer/interphase: white precipitated DNA Bottom layer: pink organic phase

Carefully remove the aqueous phase using a pipette. With a larger pipette, it is harder to control the rate and force of fluid withdrawal and this increases the likelihood of drawing some of the organic or DNA phase. Leave behind some of the aqueous phase (about 1 mm above DNA layer to prevent DNA contamination). Place in another 1.5 ml Eppendorf tube.

Add 550 µl isopropanol to the aqueous phase and mix gently. Leave at room temperature for 5 min.

Centrifuge at maximal speed (14,000 rpm) for 20 min. If a low yield is expected, centrifuge for 30 min.

Place samples on ice. There should be a pellet barely visible at the base of each tube. Pour off the isopropanol and add 1 ml 75% ethanol in DEPC treated H2O. Mix gently. Recentrifuge at 9,500 rpm for 5 min.

Pour off the ethanol and let the pellets air-dry. This is a critical step; if the pellets dry out too much, the RNA crystallizes and is very difficult to resolubilize. If not enough of the ethanol evaporates, this also prevents the RNA from going into solution. After pouring off the bulk of the ethanol wash, there will be approximately 30–40 µl left in the bottom of the Eppendorf tube. To quicken the evaporation, centrifuge the tubes briefly to force remaining fluid on the side of the tube to the bottom, then pipette off as much of the ethanol as is feasible. The best time to add DEPC treated water to the RNA pellet is when there is only a tiny meniscus of solution left around the pellet itself.

Add approximately 15–25 µl (depending on yield) of either DEPC treated TE buffer or water to the RNA pellet. To a small Eppendorf tube, dilute the RNA 1/40 (1.2 µl in 48.8 µl of TE buffer) and add to a microcuvette (path length = 1 cm). Then measure the absorbance at 260 nm. The 260/280 ratio should be greater than 1.8. If less than 1.5–1.6 or so, the RNA is likely to be at least partially degraded. Lower ratios also suggest DNA or thiocyanate contamination. The concentration is essentially the equivalent of the OD at 260 nm (in µg/µl).

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