Do you have suggestions about the preparation of RNA before miRNA RNASeq analysis? Which facility/service provider performs best the analysis and in a cost efficient way? Thanks
Hello Devrim,
Prior preparing the libraries, I would recommend you to do a proper calibration with a better technique than the usual nanodrop. If you have normal (cellular) RNA, I would recommend you to use a RNA-chip from Agilent for the Bioanalyzer to quantify and, at the same time, obtain some quality data about your samples.If you have some other weird population like I have, Ribogreen works pretty well.
It would also be good if you do also a small-RNA chip to make sure you have purified miRNAs while purifying all the other RNA populations. I tell you this because if you have used a RNA extraction kit specific to purify miRNAs (Qiagen and Exiqon have some good kits) you should have not any problem, but if you are still using old techniques like phe-chloroform and etOH precipitation you will not get the miRs (I have tested several purification methods and with the old ones we lost most of our small RNAs).
About platform and so... I prepared my own libraries for specific Small-RNA and I am very happy with the results. Also, we prepared the libraries by ourselves and so the price decreased substantially. I guess someone else can give you information about some other platforms.
Good Luck!
first please make sure what kind of small RNA do you want to catch. siRNA or miRNA? piRNA? OR all of them. usually, you should try to catch the Total RNA and
do size selection between 18bp to 25bp? Then make the library and sequence them using illumina platform. if you need data quality control, i can help you.
Shan, the SmallRNA library preparation from illumina includes a size selection after the retrotranscription. This ensures that you will not lose you RNA by degradation and the final contaminants will be much smaller. Again, I think that Devrim (I assume from the tittle) wants to look for miRs, which are the bands marked by the ladder from the illumina protocol.
Genotypic Technology at Bangalore, India is a service provider specializing in NGS.
Small RNA extraction, lib prep, Illumina sequencing and analysis can be taken care of at the company.
Its a question, not an answer t the previous query: Please reply, if you wish--
How to proceed for Annotation and Characterization of novel miRNA?
We have sequenced miRNA of bubaline/buffalo leukocytes. Some novel miRNAs have been obtained.
One example file has been attached.
We are now blasting each of the novel miRNA sequence against the miRBase which has yielded miRNA sequences from other animal species like human, mice etc (E-value as small as 0.0001). We are then opening:
1. Interaction explorer (http://www.informatics.jax.org/interaction/explorer)
2. Gene Ontology Classifier
3. KEGG
Please advice what should be the pipeline to annotate the novel miRNA in a species like buffalo where Chromosome wise genomic data are not available?
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