RNA Isolation from Bacteria

Sequencing RNA isolated from bacterial cultures is a common method for determining what genes are being expressed in the culture. Sequencing assays usually require high quality RNA. PoP team members working with a customer optimized a protocol using the RNAdvance Tissue kit for use with bacteria by the addition of lysozyme.

Protocol:

RNA isolation from Bacteria using RNAdvance Tissue

RNA Extraction from E. coli, B. subilis and S. saliverius using RNAdvance Tissue Kit

Escherichia coli, Bacillus subtilis and Streptococcus saliverius were grown overnight in LB media at 37 °C. The E. coli culture grew to an OD of 6.59, the B. subtilis culture grew to an OD of 4.15, and the S. saliverius culture grew to an OD of 0.94. RNA was extracted from 150 µL of those overnight cultures. Yield was measured by absorbance at 260nm on a NanoDrop (Thermo Fisher Scientific), and integrity was assessed on an Agilent RNA ScreenTape (Agilent). The yield of RNA correlated to the bacterial overnight OD, with higher overnight OD resulting in higher yields (Figure 1).

 

Genomics PoP RNA Isolation from Bacteria Figure 1

Figure 1

 

The resulting RNA extracted had high RIN scores, with a RIN score of 8.4 for E. coli, 9.4 for B. subtilis and 7.4 for S. saliverius (Figure 2-4) indicating highly intact RNA.

Genomics PoP RNA Isolation from Bacteria Figure 2

Figure 2

 

Genomics PoP RNA Isolation from Bacteria Figure 3

Figure 3

 

Genomics PoP RNA Isolation from Bacteria Figure 4

Figure 4

 

 

Beckman Coulter makes no warranties of any kind whatsoever express or implied, with respect to this protocol, including but not limited to warranties of fitness for a particular purpose or merchantability or that the protocol is non-infringing. All warranties are expressly disclaimed. Your use of the method is solely at your own risk, without recourse to Beckman Coulter. Not intended or validated or use in the diagnosis of disease or other conditions. This protocol is for demonstration only, and is not validated by Beckman Coulter.

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