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making a small RNA library from human serum
Hello everyone,
I am trying to sequence microRNAs from human serum using illumina. I have tried sending my sample, total RNA, to NHGRI where they made the library and then sequenced the sample. I have done this twice now using both single individuals where I average about 600ng of total RNA, and pools of individuals where i can get 1-10ug of total RNA. Both times the sequence has very few mappable reads and for the most part has not given me good data.
My question is, has anybody used any of the new kits for small RNA library prep from small amount of total RNA. I am looking at the new illumina kit, the bioo scientific air small RNA kit, as well as the ScriptMiner™ Small RNA-Seq Library Preparation Kit. Since the RNA is isolated from serum the quality is low and there are also very few ribosomal RNAs, just mostly small RNAs, degrated RNA and mRNAs, so I was not planning on size selecting first, especially since I have such small amounts of starting material.
Anyone have any experience and or suggestions for making a library from serum that will sequence better.
Thanks
Ashley
I am trying to sequence microRNAs from human serum using illumina. I have tried sending my sample, total RNA, to NHGRI where they made the library and then sequenced the sample. I have done this twice now using both single individuals where I average about 600ng of total RNA, and pools of individuals where i can get 1-10ug of total RNA. Both times the sequence has very few mappable reads and for the most part has not given me good data.
My question is, has anybody used any of the new kits for small RNA library prep from small amount of total RNA. I am looking at the new illumina kit, the bioo scientific air small RNA kit, as well as the ScriptMiner™ Small RNA-Seq Library Preparation Kit. Since the RNA is isolated from serum the quality is low and there are also very few ribosomal RNAs, just mostly small RNAs, degrated RNA and mRNAs, so I was not planning on size selecting first, especially since I have such small amounts of starting material.
Anyone have any experience and or suggestions for making a library from serum that will sequence better.
Thanks
Ashley