Hello everybody.
We have a problem with Illumina sequencing of miRNA contained in mouse serum. It is the first time we prepare a small RNA library, thus we are not able to understand where is our problem since we followed the protocol step by step. The blood was taken by cardiac puncture on anaesthetized mice.We have then isolated small RNAs using de miRNAeasy serum/plasma kit (QIAGEN) and made a library for Illumina sequencing using NEbNext® Multiplex Small RNaLibrary Prep Set for Illumina® (NEB). Before start the protocol we analyze the RNAs profiles by Nanodrop, the result were not the best, but since many articles and also the Illumina support, suggest to work in volumes because is not possible quantify exactly the smallRNA fraction, we pooled 3 serum samples of three different treatments. Each pool was made by equal volumes of serum and we assigned a different Index of the NEbNext® kit for each pool. After PCR we made a size selection with AMPure XP beads. The bioanalyzer profile showed many little peaks with a major band at 151 and 154 bp depending on the sample. We have sequenced 36 pb and found almost no miRNA. Instead, almost all the reads we have are for tRNA. Does anyone have had this problem before? Could it be the size selection with the beas was not so efficient? Is it a technical problem or it came from the bioinformatic analysis? All suggestions to resolve our problem are welcome!!
We have a problem with Illumina sequencing of miRNA contained in mouse serum. It is the first time we prepare a small RNA library, thus we are not able to understand where is our problem since we followed the protocol step by step. The blood was taken by cardiac puncture on anaesthetized mice.We have then isolated small RNAs using de miRNAeasy serum/plasma kit (QIAGEN) and made a library for Illumina sequencing using NEbNext® Multiplex Small RNaLibrary Prep Set for Illumina® (NEB). Before start the protocol we analyze the RNAs profiles by Nanodrop, the result were not the best, but since many articles and also the Illumina support, suggest to work in volumes because is not possible quantify exactly the smallRNA fraction, we pooled 3 serum samples of three different treatments. Each pool was made by equal volumes of serum and we assigned a different Index of the NEbNext® kit for each pool. After PCR we made a size selection with AMPure XP beads. The bioanalyzer profile showed many little peaks with a major band at 151 and 154 bp depending on the sample. We have sequenced 36 pb and found almost no miRNA. Instead, almost all the reads we have are for tRNA. Does anyone have had this problem before? Could it be the size selection with the beas was not so efficient? Is it a technical problem or it came from the bioinformatic analysis? All suggestions to resolve our problem are welcome!!
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