Hello to all,

This is my first time working with miRNAs and I am trying to isolate them from plasma samples. I don't know why but the service that is going to measure the miRNAs is concerning about A260/280 ratios and I have not read anything about it here. I am working with miRCURY Biofluids from Exiqon and I am obtaining 15 ng/microl with 500 microl of plasma but my ratios are about 1.6-1.7. Is this normal? Thanks!!

I used mirVana™ miRNA Isolation Kit of Ambion and miRNeasy Serum/Plasma Kit of Qiagen. As to my experience, Qiagen's kit is much better for miRNA isolation from serum. With a starting material as low as 200 ul, at least 20-30 ng/ul total RNA can be extracted. On the other hand with mirVana kit even starting with 500 ul, it is not possible to get same amounts of RNA. Moreover, with Ambion's mirVana kit, the concentrations you obtain is much more variable from sample to sample. 

Thank you Omer for sharing your opinion. I had good experience with miRNAeasy Serum/Plasma kit (cat. No. 217184 Qiagen), however I did some modifications.

Firstly, I used Trizol LS Reagent (cat, No. 10296-028 Ambion) instead of QIAzol Lysis Reagent.

Secondly, to obtain more microRNA or plasma (or serum) I used 300 uL of pasma instead of 250 uL (as was recommended).

You said that you finally got 20-30 ng/uL of total RNA. How did you evaluated this amount? From my experience, I used Eukaryote Total RNA Pico chips, I finally had average concentration of 60 (SD 32) pg/uL. This amount is more or less confirmed by Small RNA chips as miRNA concentration.

Thank you Omer for sharing your opinion. I had good experience with miRNAeasy Serum/Plasma kit (cat. No. 217184 Qiagen), however I did some modifications.

Firstly, I used Trizol LS Reagent (cat, No. 10296-028 Ambion) instead of QIAzol Lysis Reagent.

Secondly, to obtain more microRNA or plasma (or serum) I used 300 uL of pasma instead of 250 uL (as was recommended).

You said that you finally got 20-30 ng/uL of total RNA. How did you evaluated this amount? From my experience, I used Eukaryote Total RNA Pico chips, I finally had average concentration of 60 (SD 32) pg/uL. This amount is more or less confirmed by Small RNA chips as miRNA concentration.

Another thing which should be considered is an elution step. The elution volume definitely treats final miRNA concentration. What I do usually, I elute spin column with a small volume of RNAse free water (14 uL), but I do it twice to two separate collection tubes. This way I have almost twice more miRNA. In the second elution I usually have similar amount of miRNA.

In case of miRCURYTM RNA Isolation Kit by EXIQON I elute in 25uL of RNAse free water.

There are bias for particular RNA subsets with all kits means each kit have certain chemistry that will provide specific sets of RNA and miss many ....kindly find the link.

miRCURY RNA Isolation Kit could be good option to start .

http://www.sciencedirect.com/science/article/pii/S0161589012000259

I actually quantified with nanodrop, which I guess doesn't give 100% sure concentration data, however, to use in qRT-PCR experiments I think it is more than enough.  

Ganesh, I can agree with you. Now I use miRCURY kit for plasma and my expression profiles look better.  As far as assessment of miRNA concentration by Nanodrop, I cannot recommend this method as a gold standard for miRNAs measurement. 

Other question about plasma or serum miRna quality is a preanalitical phase. Sample probing can influence seriously muRna quantity and profile.  I mean hemolysis for example. 

We've had pretty good success with following the mirVana Paris kit protocol, using the modifications published in this article:

http://rnajournal.cshlp.org/content/19/5/712.long

Burgos, et. al., "Identification of extracellular miRNA in human cerebrospinal fluid by next-generation sequencing" RNA 2013. 19: 712-722  ( doi: 10.1261/rna.036863.112 )

I had great results with TaqMan ABC Purification kit produced by Life Technologies (Ambion). this kit works with magnetic beads and it's very easy!

I have another question: which is, according to your experience, the best circulating miRNA reference control for plasma or serum samples?

Dear All,

Thank you very much for your suggestions. Sorry for my absence.

Thank to Jay for the paper by Burgos et all. (RNA 2013). It is just what I was looking for last days. I am about extraction from body fluids for NGS. Great!

As far as “the best circulating miRNA reference control for plasma or serum samples”. It is very important issue for me, and I have some experience.

In my research I used miR-16 as a reference clinical plasma samples in patients with cardiovascular diseases. These data are under revision now, and I cannot send you the manuscript. Many people use it as a reference for solid tissue, and cancer. I found only ONE experimental paper about miR-16 as a reference in serum:

http://www.ncbi.nlm.nih.gov/pubmed/22198701

Song et al. Identification of Suitable Reference Genes for qPCR Analysis of Serum microRNA in Gastric Cancer Patients. Dig Dis Sci. 2012 Apr;57(4):897-904. DOI 10.1007/s10620-011-1981-7

In my other patient group, with acute pancreatitis, I found some discrepancies in the miR-16 expression among patients. In my control group, I found stable upregulation of miR-16 and miR-103 using global normalization approach (in compare to selected miRNAs which I used as markers of disease). However, in acute patients, both miR-16 and miR-103-3p were up- or down regulated, depending on the state of a disease.

Unfortunately this paper is also under revision, so I cannot show you my data.

From the other side, not only a patient state is important in miRNA profiling. You must be aware of so called “the preanalytical phase”. The sampling conditions (are platelets activated during sampling!), sample preparation and storing. Especially whole blood if very “dynamic matter”. You must avoid hemolysis. Exiqon for example recommends miR-451a and miR-23a-3p as unique hemolysis indicators. In my experience, the simplest, but not the best, is the Harboe direct spectrophotometric method: A 3-point absorbance measure (415, 380 and 450 nm)

Han et al. Han A_comparative_study_of_common_techniques haemolysis in stored red cell concentrates 2009. DOI: 10.1111/j.1423-0410.2009.01249.x

http://onlinelibrary.wiley.com/doi/10.1111/j.1423-0410.2009.01249.x/pdf

Ewa

Thank you very much, Ewa! I will try with miR-16 and I will update you after my experiments

I usually extract miRNAs from biofluids (generally plasma) with comercial kits. Nowadays I'm using miRCury miRNA extraction kit (Exiqon). I apply different quality control points. First, I measure all samples in nanodrp to check for hemolysis and discard those that don't pass this firs step control. For normalization purpose, I use a mix of spike in that I add to the sample before the extraction (but after the addition of the lysis buffer which contains RNase inhibitors, otherwise, the spike in will be deraded).

Dear all,

I am trying to extract miRNA from plasma samples. I am currently using serum /plasma miRNA esasy kit from Qiagen. I start with 200uL of plasma and I end up with 20-150 pg/uL RNA concentration. For downstream applications, I will need around 2ng /uL RNA concentration.

I saw on previous post that Ewa had a similar RNA final concentration.

Does anybody knows how to fix this issue ?

Thank you for your inputs.

Thank you,

Hi Mathieu!

I have extracted miRNA from human plasma samples (200uL) with miRNeasy (without spike in) adding glycogen to better recovery, following instructions from: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3655275/

The concentration of samples is about 8,5ng/uL on average, using Qubit miRNA assay. Maybe you should try to do it.

Hi Mathieu,

From the little detail you've written, it looks as though you need to have 100 X concentration.  What is the total mass (how many picograms) of RNA you are able to isolate from 200uL?  I have not seen any nucleic acid isolation from plasma that will yield as high of a concentration as 2ng/uL, but you can concentrate the isolated RNA.  We use the Zymo Research Clean & Concentrator 5 columns with pretty good results.  However, I suspect that you will need to increase the volume of plasma from which you are isolating the RNA to get the total amount you need for your application.

Hi Mathieu,

We use miRCury RNA Isolation kit - Biofluids (Exiqon)

We obtain about 10 ng/µL but including spike in from 200 µL

Thank you all very much for your inputs

My latest analysis of clinical serum from acute pancreatitis patient reveled that a recommended hsa-miR-16-5p was not stable expressed in our samples. We observed significant fold change (RQ: 6.4 (p=0.045)) in mild AP and severe AP vs. controls (RQ: 28 (p=0.03)). In opposite to miR-16, there was no significant variability in the hsa-miR-103a-3p expression in those patients vs. control (RQ: 1.1 (p=0.454) and 0.9 (p=0.099), respectively) as well as in MAP vs SAP patients (RQ: 0.8 (p=0.291)). My interpretation of this finding g is that in these particular patients we always observe: inflammation, vascular complications and organ injury (on different levels). Those conditions can influence miR-6 over-expression and its release to circulation.

Many thanks Ewa! For my studies I need of one or more miRNA stabily expressed in plasma of healthy subjects only. I will try with miR-16, miR-191 and miR-93.

Dear all,

with this post I would to update you about my experimental research. In all healthy plasma samples that I used, I found both miR-16, and miR-93 and miR 191! And the real surprise is that with our workstation (OMNIA Prima by MASMEC Biomed) the yield of extracted miRNA is higher than manual extractions!

dear all,

I used Qiagen serum/plasma kit for extraction of miRNA from human plasma (100ul). i used the spike in. Once eluted the RNA, i added 1,5 ul in the cDNA synthesis reaction. i diluted cDNA 1:2 and in the end I couldn't detect signal below Ct 34 at the qPCR. any suggestion?

thank you in advance for your help! 

Hi  Monika, the RT-PCR before qPCR was specific for the spike or for total extraxted RNA?

Hi Monika, to be honest, I've not found the most appropiate protocol yet.

I've used both, miRneasy mini Kit serum/plasma (Qiagen) and miRCury extraction kits biofluids (Exiqon).

With Qiagen's protocol I usually obtain higher amonts of miRNAs but I hav problems in the amplification because the Non-template control amplifies at cycles similar (or very close) to the samples.

With Exiqon's protocol Cts are higher.

Another problema I have is the variability between experiments (both biological and technical replicates) in terms of Cts

Dear all, What is the best method to extract RNA from plasma if it is needed for sequencing? We've used the Qiagen miRNeasy kit but the yields are quite simply too low (we require 50ng/ul). Any suggestions??

Hello to all,

This is my first time working with miRNAs and I am trying to isolate them from plasma samples. I don't know why but the service that is going to measure the miRNAs is concerning about A260/280 ratios and I have not read anything about it here. I am working with miRCURY Biofluids from Exiqon and I am obtaining 15 ng/microl with 500 microl of plasma but my ratios are about 1.6-1.7. Is this normal? Thanks!!

Dear Isabel,

I use the same kit to purify microRNAs from plasma. For my experience I think it is the best kit. But I obtain similar 260/280 ratios, which is common when working with that kind of samples that yield low miRNA concentrations

Hi Lidia, we were together the other day. Thanks for your information, I am now more confident with my work. 

Hi Isabel,

Yes, I remember the work you presented in the last SEA congress as we presented our Works in the same session. I told you then, but again, nice presentation!

Regards,

Lidia

Hi

I found this nice paper regarding the Endogenous Control Selection for serum exosomal miRNA qRT-PCR

Validation of Isolation Methodology and Endogenous Control Selection for qRT-PCR Assessment of Microrna Expression in Serum and Urine Exosomes

Rachel E Crossland, Jean Norden, Louis Bibby, Joanna Davis, Anne M Dickinson

Blood 2014 124:5793;

Hello

Can any one tell me how to isolate microRNA from platelets. if possible please provide protocol.

Thanks in advance.

Dear sir ,

The  question relates to MEDICAL SCIENCE  and not to ion plasma.

I am sorry ,I have no answer.

R.G.Rastogi.

 Hello all,

I was reading through the posts because I am myself struggling with RNA extractions lately. I am extracting total circulating RNA from 500 µl CSF and 300 to 500 µl plasma using the mirVana PARIS kit. My main problem is that centrifuging after the addition of phenol:chloroform doesn't give me a nice white interlayer. However, after extracting the upper layer and adding the same amount of nuclease free water back to the remaining lower phase and centrifuging again, I obtain said interlayer. I have tried incubating my samples longer before centrifuging, and centrifuging for a longer time at different speeds, but the same happens every time. I elute in 50 µl of  water, do a DNase treatment, and measure the concentration on a Nanodrop to have a first quality check. I obtain  low concentrations (< 10 ng/µl) and very low 260/280 and 260/230 ratios. The only step where something could go wrong, I think, is during the phenol:chloroform extraction. Has anyone experienced the same with this kit or do you know what else I could try?

Thank you very much!

Best,

Clara

Dear all,

Few years ago, as many of you, I used miRCURY Biofluids from Exiqon for extracting RNA from plasma for detecting miRNA by qPCR. However, this product is currently discontinued since Exiqon is now integrated with Qiagen. So from your latest experience, what is the best method to extract RNA (including miRNA) from plasma if it is needed for NGS RNA sequencing? do you use a kit for extracting only miRNA such as NucleoSpin® miRNA Plasma (MACHEREY-NAGEL)? or do you use a kit for extracting the total RNA such as miRNeasy Serum/Plasma Advanced Kit (QIAGEN)?

I am wondering if you use the total RNA for NGS sequencing, high proportion is mRNA, have you any idea/experience how that could affect to the sequencing of the small RNAs?

Thank you,

Best,

Salam