Bioanalyzer for microRNAs has never worked really well (i tried the specifically designed one for small species). I think i can easily send you a picture of a normal chip, that show the mess of degraded species typically present in plasma and serum, that suggest NOT to quantify. If you have to profile, go for equal VOLUMES, and work out a strategy of normalization. I add also that pathological conditions are sometimes characterized by increased exosome level and global amount of microRNAs in serum (eg cancer). For normalization maybe you can refer to the paper we published on embo molecular medicine, concerning serum microRNA for diagnostic purposes in lung cancer.

We can start with 200microliter

The mininal volume to start with should be 200 microliter

Hi Andrea

We routinely extract total RNA from serum using 300ul as starting point. The protocol that we employ is based on the described by Exiqon. First a phenol extraction with Trizol LS (more concentrated than normal Trizol to extract from liquid samples) and after that a column purification in a RNAeasy kit.

Do not worry about the concentration... it is normal. The yield is typically in the range of a few nanograms.

Let me know if you need more details.

Good luck. Cheers. Paco

the starting volume should be more then 400 microliter other wise the volume of RNA will be to low for quantification by spectrophotometer so the mininum of serum taken be increased

Tewari's methods works perfectly!

and suggest addition of spicked in oligos for recovery of RNA

the volume to start with is very low you should start with a higher volume and at the same time it should be higher then 400microliter can follow the modified protocol from this paper in method

Analysis of circulating microRNA biomarkers in plasma and serum using

quantitative reverse transcription-PCR (qRT-PCR)

Evan M. Kroh

a

, Rachael K. Parkin

a

, Patrick S. Mitchell

a,1

, Muneesh Tewari

a,b,c,

*

I would suggest 1) trying different starting volumes and 2) maybe decreasing your elution volume at the end so you get a more concentrated sample. We have tried it with 1mL of plasma in 4mL of Trizol LS with an final volume of 12ul giving us a concentration of anywhere between 25 to 100 ng/ul. We always use glycogen as a carrier for our isopropanol precipitations and we do not use Qiagen's RNeasy kit for clean up if we want to detect miRNAs (they are not large enough to bind to the column).

or else do start up with a trizol method up till the isolation the recovery part can be done by using purification coumns from other kits like mir VAN as you tried the other kits and you can also use some spike in for better recovery..

or the a good kit for isolation of miRNA from NORGEN but here the sample size has to be increased from 200microliter to around 2ml to 5ml

Fimal elution volume is 100 microliter but the the sample size is more so in order to decrease the sample volume we can use trizol in combination with the kit

Concentrate circulating RNA – Circulating RNA present in input volumes of 0.25 - 5 mL are concentrated into final elution volume of 100 µL.

I use the norgen slurry kit and am able to load 1ml of plasma, the conc ranges from

as low as 2-30ng/ul depending on the sample but this is eluted in 100ul so am able to dry down sample to to concentrate, I always do a second elution into 50 ul and often this is more concentrated than the first

we can isolate the toral RNA using guanidine isothiocyanate for total RNA isolation

Total RNA to be isolated precipitated overnight to increase the yield purification step to be performed using Column from kit

protocol adapted from

Adapted from the Instruction manual of Stratagene's Micro RNA Isolation Kit (Catalog #200344)

There are two main methods for isolating RNA: phenol-based extraction and silica matrix or glass fiber filter (GFF)-based binding. Phenol-based reagents contain a combination of denaturants and RNase inhibitors for cell and tissue disruption and subsequent separation of RNA from contaminants.

Phenol-based isolation procedures can recover RNA species in the 10–200-nucleotide range (e.g., the miRNAs, 5S rRNA, 5.8S rRNA, and U1 snRNA). If a sample of “total” RNA was purified by the popular silica matrix column or GFF procedure (i.e. Qiagen RNEasy columns), it will be significantly depleted in small RNAs. Extraction procedures like Trizol/TriReagent, however will purify all RNAs, large and small, and are the recommend methods for isolating total RNA from biological samples that will contain miRNAs/siRNAs.

This protocol utilizes the powerful guanidine isothiocyanate–phenol:chloroform extraction method which allows the rapid isolation of RNA. The conditions for extraction enable the partitioning of proteins and DNA into the organic layer of the biphasic solution interface, while retaining RNA in the upper aqueous layer. The aqueous phase is removed to a second tube, and RNA is precipitated with an equal volume of isopropanol. High yields of pure, undegraded total RNA can be recovered from even small quantities of tissue or cells. Large numbers of samples may be performed simultaneously, because of the simplicity of the technique.

There are two main methods for isolating RNA: phenol-based extraction and silica matrix or glass fiber filter (GFF)-based binding. Phenol-based reagents contain a combination of denaturants and RNase inhibitors for cell and tissue disruption and subsequent separation of RNA from contaminants.

Phenol-based isolation procedures can recover RNA species in the 10–200-nucleotide range (e.g., the miRNAs, 5S rRNA, 5.8S rRNA, and U1 snRNA). If a sample of “total” RNA was purified by the popular silica matrix column or GFF procedure (i.e. Qiagen RNEasy columns), it will be significantly depleted in small RNAs. Extraction procedures like Trizol/TriReagent, however will purify all RNAs, large and small, and are the recommend methods for isolating total RNA from biological samples that will contain miRNAs/siRNAs.

This protocol utilizes the powerful guanidine isothiocyanate–phenol:chloroform extraction method which allows the rapid isolation of RNA. The conditions for extraction enable the partitioning of proteins and DNA into the organic layer of the biphasic solution interface, while retaining RNA in the upper aqueous layer. The aqueous phase is removed to a second tube, and RNA is precipitated with an equal volume of isopropanol. High yields of pure, undegraded total RNA can be recovered from even small quantities of tissue or cells. Large numbers of samples may be performed simultaneously, because of the simplicity of the technique.

For this protocol the starting material can be as low as 50 microliter and the yield is compartively higher have tried for 100 microliter and the yield is as good as 40 microgram.

The equation to be used 0.10

spec. reading at A260

× 500/5

Dilution factor

× 0.04 μg/μl

Conversion factor A260

= 0.4 μg/μl

Final concentration

In the protocol discussed below the starting volume can be as low as 50microliter and the yield can be as high, for 40 microgram for 100 microliter of your sample taken...

May be, you can prove with 200, 300, 400 µl for slope

I also use 100 µl plasma for RNA extraction. I use the Norgen Total RNA Purification kit, from Norgen Biotek. As I have understood it the levels of miRNA in plasma/serum are very low, and they are usually not detectable with for example NanoDrop (measures down to aprrox. 2 ng/µl). This was the case also in my study. In this case it is better to always take the same volume for cDNA synthesis instead of a certain amount. It is important to include control miRNAs, for example C. elegans miRNAs that is added to the lysis buffer before RNA extraction.

Hi Andrea,

I think 5 ng/µL is actually good and not really that bad. You should expect less than that!! 100 µL of cell suspension would not be expected to generate amounts very far from that.

It also depends on the downstream applications you plan to run, If you are planning to run reverse transcription followed by qPCR, then there should be no problems if you have something in the range 1-5 ng/µL.

Good luck!

Majid

sorry guys but the quantification of RNA extracted from plasma and serum CAN NOT BE DONE with nanodrop or similar instruments. Have you aver tried to look at the rna using a bio-analyzer? It is fully degraded. Only MicroRNAs are stable in plasma and serum, but their amount is very small compared to RNA species usually quantified at 260nM. Thus, i strongly suggest to:

1) Isolate microRNAs with a combination of TRIZOL LS and column-based purification kit (such as the miRNEASY by QIAGEN). 100 uL are usually enough, but try also 200 and 300 and measure your recovery (see 2)

2) measure the recovery by quantification (RT-qPCR) of some microRNAs highly expressed in the plasma, or medium expressed or low expressed.

Please note also that PLASMA IS VERY TRICKY as a biological source, since it is very easy to have contamination by platelets that also contain microRNAs. If you go for clinical applications, i think serum is better.

hi and good luck

Matteo

Only few minutes ago I was able to read all of your messages (a huge number, actually).

First of all thanks to all.

I have some considerations to share with all of you.

- I appreciate the suggestion to use Trizol LS (more concentrated than classic one). I will try both versions.

- I do not agree completely to use RNEasy kit for miRNAs recovery, instead I do not know how the miRNEasy works. Maybe better according to manufacturer. I have alternative spin column kits to try (miRVana or Norgen) that I expect they perform better than Qiagen ones.

- Tewari's protocol is good, although it has been reported only the use of Qiazol.

- Question for Mariano: have you tried also to use spin columns?

- I am not particularly convinced that spike-in controls are a good option for downstram applications, as many mature miRNAs can share the same sequence with other human miRNAs or interfere with hybridisation (if you want to perform an array profiling). What is your opinion?

- For Camilla Skoglund and Bram van der Eerden: did you follow the Norgen protocol to extract your sample?

- Concentration of approx 5 ng/µl is good for qPCR but not for profiling where the amount should be more than at least 150 ng. I hope that Christopher Eicken could see this message and let me know his experience on miRNAs extracted from serum or plasma and the amount to use for miRNA profiling.

- For Matteo: it would be great if you can send me an image (or the 2100 BIoanalyzer data file) where you quantified your miRNA sample to see how the fraction of small RNA looks like. However, I need a quantification to perform profiling, and yes, maybe the choice of serum is preferable. Have you compared the two (serum and plasma?). Some manufacturers suggest to use plasma....

Bioanalyzer for microRNAs has never worked really well (i tried the specifically designed one for small species). I think i can easily send you a picture of a normal chip, that show the mess of degraded species typically present in plasma and serum, that suggest NOT to quantify. If you have to profile, go for equal VOLUMES, and work out a strategy of normalization. I add also that pathological conditions are sometimes characterized by increased exosome level and global amount of microRNAs in serum (eg cancer). For normalization maybe you can refer to the paper we published on embo molecular medicine, concerning serum microRNA for diagnostic purposes in lung cancer.

Matteo, I also tried the Small RNA kit from Agilent and I quite agree with you about results. I am curious to see the image, if you can send it to me. The point of using volumes instead of concentration, is that I need to pool samples coming from different patients and I prefer to pool quite the same amount. At least an indication of the amount in each sample is desirable not to produce artifacts...I do not know anything of these samples, so normalization using some miRs is a sort of "try ans see" at least at the beginning. I will read your paper and I will let you know if in doubt.

Thank you.

Andrea

I agree with the nanodrop comments, we find that concentrations below a certain amount (~50ng/ul) are not accurate. Our concentration readings from the Agilent Bioanalyzer are more accurate.

Dear all, if you look at the isolation of miRNA from low content samples like serum you should also be aware of the following thread: https://www.researchgate.net/post/RNA_extraction_using_10k-50k_cells

and the discussion regarding the Kim et al. publication.Molecular Cell 46, 893–895, June 29, 2012 basically they publish a caveat that GC content in low RNA concentration samples will affect the extraction and introduces bias when using phase separation extraction with trizol. So as a minum the extraction amount is to be held constant, or carrier should be used in addition.

I think the quantification of RNA extracted from plasma and serum CAN NOT BE DONE with nanodrop or similar instruments. Have you aver tried to look at the rna using a bio-analyzer

Quantification of RNA results only when we add bacterial RNA to the process.

Hi,

For extraction of serum/plasma RNA, if at least 100ul of serum is available, I tend to use miRVANA PARIS kit (have tried miRNA Easy (Qiagen)- didn't work well, standard Phenol:Chloroform protocols- worked as good as miRVANA, and Trizol LS- more variability than column extraction). I quantify by Qubit (Invitrogen) ( NanoDrop- not suitable). The levels of RNA I recover from 100ul of mouse serum range from 50-500ng, much lower for human serum.

Both my research lab at Ghent University and Biogazelle's service lab have been happy users of Qiagen's miRNeasy kit (mini and micro version) for total RNA purification (including microRNAs) for several years. We process tissues, cultured cells, serum, plasma, sputum, saliva, etc. We typically start with 200 µl of serum, but 100 µl or smaller volumes also work (at a cost of lower detection rate).

What i ve read for few days that i'm on research gate is that everyone has is own preference. Some say "this one is good" and the other says "it didn't work" talking of the same kit... well for me who is a beginner, it is very hard to know which kit i should try. And above all, as each kit is very expansive it s just a hard chineese puzzle to chose the kit I'll use!

I will have approximatively 100uL of mouse plasma and i would like to isolate miRNA. I have in my lab some mirVana miRNA Isolation kit, some miRCury RNA Isolation kit-Biofluids and some Tripure. In those 3, only the miRCury is recommended for plasma. Do you think that the other 2 could be used for plasma? (I know that some people are using the miRNeasy mini kit for plasma in place of the miRNeasy kit for plasma/serum. Maybe it could be the same for the mirVana?)

what is really the difference between all those kits?

dear Floriane, your feeling towards different comments on RG is quite natural! you have to try several ways to find the most proper method for your experimental conditions and don't be afraid for this is research! about your question, Trioure is not a suitable reagent for Plasma miRs isolation because it is commercially provided in low concentration that makes it unsuitable for liquid samples. if your kit says: do not use for liquid samples it means that they cant recover small RNA fragments (which is predominant in Plasma and Serum) or again their first reagent is not provided in high concentration.