Dear Maulik,

It depends what kind of samples you plan to analyse. If you plan to use plasma or serum samples, microarray methods are not recommended, becase of yld of miRNa in such samples. The same is urine or other body fluids.

In such case I recommend you qPCR methods, some companies offer such approach for miRNA profiling. You can aslo consider NGS, in this case you have both quantitative and qialitative analysis.

Ewa

@ Ewa Stepien

How much yield required for MicroArray analysis, if I isolate MicroRNA from blood samples ?

I am planning for go for customize microRNA MicroArray for about 6 - 10 highly specific probe only, based on R analysis. Mainly looking for Breast Cancer.  

Hi,

For microarray analyzes you usually need at least 1 µg of total RNA and the quality is limiting. In the case of bloos samples, it is usually needed to make a pool of different samples to reach the required amount.

If you are planning to analyze onli a list of speciffic miRNAs, you can use customized miRNA PCR plates or TLDA cards.

Regards

@ Georgia Makrypidi

Thank you sir for your answer. 

I am working with Breast Cancer & Oral cancer, where many MicroRNAs are common, also some microRNA can target more than 2 cancer. With all bioinformatics analysis, i found out some microRNA which are up-regulated for Breast cancer, Down-regulated for Breast Cancer,  up-regulated for Oral cancer, Down-regulated for Oral Cancer.

Out of those, some microRNA are UP & Down both in different stage of same cancer.

I analyzed all those data and found out list of 10-15 microRNA, for those, i am planning to go for MicroArray studies. 

Please guide me, if anything missed out or some point that i need to keep in mind.

thanking you,

Maulik Rachh

@ Lidia Daimiel-Ruiz

Dear Mam, thank you for your answer. 

I am isolating microRNA from Blood sample only, and planning to study for Real Time qPCR for identification purpose.

But before that, i as mention above answer, study which may be useful for me and give me more confidante for the same.

please guide me if any thing wrong or not in proper way.

thanking you,

Maulik Rachh

Are you planning to analyze miRNAs in total blood or in plasma/serum?

@ Lidia Daimiel Ruiz

Yes i am planning to isolate miRNA form Whole Blood. 

@ Lidia Daimiel Ruiz

Dear Mam, can you please add some more information about " TLDA Cards "? 

Means if i have list of 10 microRNA for analysis, so how to go with TLDA cards analysis?

@ Georgia Makrypidi

Dear Mam,

Thank you for your reply and sorry for mistake.

For analysis of the microRNA, I used HHMI MicroArray data, and analysed using R for Heatmap Generation. I found list of top 10 microRNA which are UP/Down Regulation for Breast Cancer & Oral Cancer. 

If I am not wrong, Up-regulation microRNA which are coming from Oncogenes and Down-regulation MicroRNA coming from Tumour Suppressor genes. For Diagnosis purpose Up regulated MicroRNA will be more useful.

MicroRNA downregulation is the reduction in the quantity of small non-coding double-stranded RNA molecules called MicroRNAs that are critical in the regulation of gene expression.

That's why i am planning to go for MicroArray analysis for gaining more detail about the same.

Thanking you,

Maulik Rachh  

Dear Maulik,

First of all, in my personal opinion, I don't think that analysis of miRNA from blood samples of patients suffering from oral/breast cancer or any other solid malignancy is a good idea. This is because that blood cells are not the malignant cells involved so you are not going to get any significant results. It would have been better to collect biopsy or surgically removed cancer tissues and adjacent healthy tissues and compare the expression profile of miRs using any of the available microarray platform and further validate the expression of only dysregulated miRNAs using qPCR. Otherwise, you could have collected saliva from oral cancer and nipple aspirates from breast cancer subjects. Still, if you are unable to get these samples, then the plasma/serum samples will be more useful as tumor derived circulating nucleic acid as well as miRNAs released from circulating tumor cells (CTC) and micrometastatic tumor cells have been reported to be present in these body fluids. 

Once you get dysregulated miRNAs, then you should look for their target genes using at least 2-3 different bioinformatic miRNA target prediction tools and see if these target genes are involved in any metabolic or signaling pathways previously implicated in carcinogenesis. If you find a good target, it would be advisable to correlate the expression of that miRNA along with its target gene in the same sample. This would definitely give you a good insight on the pathogenesis of disease.

All the best.

Sachin

Dear Mulik,

I agree with Sachin Kumar. If you use total blood, your results will correspond to the expression profile of miRNAs in blood cells, which are not the malignant cells. For that reason, it is better to use plasma/serum samples to analyze the profile of circulating miRNAs in patients and healthy subjects or to correlate the profile of circulating miRNAs in different stages of the disease. By this way, you can describe new biomarkers of disease stage and progression. At this point, some reports advise to use plasma rather than serum samples because the quality of the yielded miRNAs seems to be better in plasma.

If you aim to perform a profile of miRNAs, you could use microarrays. After that, you can select the differentially expressed miRNAs (I advise you to use a very stringent cutoff couple to multitesting corrections such as Bonferroni's correction or False Discovery Rate to avoid false positives) and validate the expression using PCR.

To look for putative target genes of the selected miRNAs, I usually use miRWlak that allow you comparing different algorithms and I select only those targets detected by, at leats, 5 algorithms with stringent perdiction criteria.

I hope this information would be useful

Regards,

Sorry but I desagree with both collegues that suggest no association of solid tumor with plasma or serum profile to have change to find out new biomarker.. It is important to consider that miRNA can be transported in microvesicles, that are present in serum or plasma. We all know that miRNA can be delivered to plasma and be detected by real time PCR as many paper have suggested. The limitation of microarray of microRNA is the sequence spoted there is already known. The better way is to make NGS (miRNOMA profiel that you can find out new sequence not described. So in my oppinion this profile can be validated by real time PCR in sample of patients with the same suspected disease, in this case cancer.

Hi Mario

You have a pont there. If the purpose is to discover new unknown miRNAs, NGS is the method.

Mulik, if you want to analyze the expresion of yet known miRNAs, then you can use microarrays that contain probes to cover all known human miRNAs (according to the latest versions of miRBase)

Regards,

@ Lidia Daimiel Ruiz

Thank you for your reply. Information provided by you is very much useful.

Still one question in my mind is that if we go for MicroRNA Sequencing as you mention for finding out Novel MicroRNA.

Characteristic small RNA biogenesis processing patterns are used for the discovery of novel microRNAs (miRNAs) from next-generation sequencing data. But how to conclude that that unique signature which we found based on analysis is MicroRNA ?

Means if i am not wrong same MicroRNA represent more than one cancer sometimes.

To identify that, it is important to run large numbers of samples with each stage of the cancer, and more than one cancer.

Please guide me its right or wrong?

Thanking you,

Maulik Rachh 

@ Sachin Kumar

Thank you sir for your answer.

It is not possible that we isolate whole RNA from Blood will give you sRNA & miRNA also? If i am not wrong several kits are available for MicroRNA isolation form Blood. 

@ Mario Hirata

Thank you for sharing information.

Dear Maulik,

Effectively, you can carry out expression analysis by comparing the runs of the same sequence in different samples to conclude that a miRNA is upregulates or downregulated in one sample.

There are several algorithms that allow you to identify if a sequence correspond to a putative miRNA. Different algorithms use different marks, including the ability to form stem-loop estructures and the complementarity between the putative guide and passenger sequence.

But, as you said, you need to process many samples. NGS costs have been reduced last years, but this technique is still unafordable for many samples. Then, you may better use microarray.

Regards,

@Mario, Please read my observations again. I have clearly mentioned that miRNAs will be present in serum or plasma. My apprehensions is about measuring miRs expression from RNA isolated from blood cells which are not true representation of malignant tumor cells in solid malignancy. NGS is quite a costly technology in India and can cost anywhere between 1000-2000 USD per gene...although you can get novel information which is not possible by microarray as it probes for known miRs only.

ncRNA are very important in terms of adding complexity to life .The mistries are still very darken about their regulation

A key point is the putative role of microRNAs as biomarkers of different diseases. To detect new biomarkers, microarray profiling is the best approach