Total RNA were extracted from bacteria grown to the middle of exponential phase (OD600nm = 0.5) using RNeasy Protect bacteria Mini Kit system (Qiagen) according to the manufacturer’s instructions. cDNA synthesis was performed from 1 μg of total RNA with random hexanucleotides as primers using the Superscript IV First Strand Synthesis system for RT-PCR (Invitrogen). The quantitative PCR reactions were then carried out with the appropriate primers (Supplementary Table 4 ) using DyNAmo ColorFlash SYBR Green qPCR kit (Thermo Scientific) and they were run in a StepOnePlus Real-Time PCR system (Applied Biosystems). The data were analyzed with StepOne software v2.3 using ΔΔCt method and normalized using the housekeeping genes rrsA, gyrA and ffh as reference genes.
Rneasy protect bacteria mini kit system
Manufactured by Qiagen
The RNeasy Protect Bacteria Mini Kit system is a laboratory equipment designed for the isolation and purification of high-quality total RNA from bacterial samples. The core function of this kit is to efficiently extract and purify RNA from a wide range of Gram-positive and Gram-negative bacteria.
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2 protocols using rneasy protect bacteria mini kit system
1
Bacterial Transcriptome Analysis via qPCR
2
Bacterial Transcriptome Analysis via RT-qPCR
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Total RNA were extracted from bacteria grown to the middle of exponential phase (A600nm = 0.5) using RNeasy Protect bacteria Mini Kit system (Qiagen) according to the manufacturer’s instructions. cDNA synthesis was performed from 2 µg of total RNA with random hexanucleotides as primers using the SuperScript IV First Strand Synthesis system for RT-PCR (Invitrogen). The quantitative PCR reactions were carried out using DyNAmo ColorFlash SYBR Green qPCR kit (Thermo Scientific) and were run in a StepOnePlus Real-Time PCR system (Applied Biosystems). The data were analyzed with StepOne software v2.3 using ΔΔCt method and normalized using the housekeeping genes rrsA, gyrA and ffh as reference genes.
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