Qiagen
RNAprotect Bacteria Reagent (2 x 100 ml)
76506
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I used Qiagen's RNAprotect Bacteria Reagent to stabilise RNA prior to extraction step so that whatever the gene expression measured after extraction should not be contributed from the factor during extraction steps. Quality of RNA samples extracted with or without RNA protect reagent were assessed by Agilent Bioanalyzer and as well by qPCR experiments. Agilent Bioanalyzer analysis revealed very good RIN numbers for the samples which were extracted after stabilising with RNA protect reagent when compared to the samples extracted without stabilisation. qPCR experiments revealed inconsistent gene expression pattern (internal reference gene) in the case of samples without stabilisation.
Extraction of very high quality RNA samples for qPCR and RNA-seq experiments
3 ml of Salmonella enterica cultures grown to an OD600 of 2.0 in LB broth
RNA bacteria protect to bacterial cultures were used at the ratio of 2:1 as per kit's instructions. Briefly 6 ml RNA protect reagent and 3 ml of bacterial culture was mixed well and allowed to stand for 30 minutes at room temperature. During this period total RNA in the sample will be stabilised. Then pelleted the mixture at 4000 rpm at 4 degree C for 10 minutes. Then followed extraction steps according RNeasy Midkit protocol. Finally eluted total RNA in nuclease free water. Samples were then used for Agilent bioanalyer analysis and qPCR experiments.
None
Agilent bioanalyzer analysis gave RIN values of 9.2 to 9.5 for the samples extracted using RNA protect Bacteria reagent stabilisation. Whereas samples extracted without stabilisation gave RIN values of 7 to 7.5. Furthermore gene expression of internal reference gene (csrA) in the samples extracted without stabilisation revealed altered expression pattern as opposed to no change in the expression pattern expected for internal reference gene.
Simple and easy to use
Stabilisation of RNA using Qiagen RNA protect bacteria reagent is a must for total RNA extraction and the RNA samples intended to be used in qPCR and RNA-seq experiments.
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