QuantiTect Reverse Transcription Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Incubate for 2 min at 42°C. Then place immediately on ice. Add reverse-transcription master mix and incubate for 15 min at 42°C. Incubate for 3 min at 95°C to inactivate Quantiscript Reverse Transcriptase

Protocol tips
Incubate for 2 min at 42°C. Then place immediately on ice. Add reverse-transcription master mix and incubate for 15 min at 42°C. Incubate for 3 min at 95°C to inactivate Quantiscript Reverse Transcriptase

Publication protocol

Bacterial cells were stabilized with RNAProtect Bacteria Reagent (QIAGEN) and total RNA extracted from 500 μl early exponential growth phase cultures and 250 μl stationary growth phase cultures using RNeasy Mini Kit (QIAGEN) according to the manufacturer’s instructions. A 15 minute on-column DNase digestion with RNase-Free DNase (QIAGEN) was performed to ensure removal of contaminating genomic DNA, as suggested by the manufacturer. For each strain, three RNA extractions were performed from exponential and stationary growth phases, while two extractions were performed for the iron-depletion experiment. RNA concentration and purity, determined by 260/280 and 260/230 ratios, were measured with a Nanodrop ND-1000 spectrophotometer (Nanodrop Technologies Inc.). RNA integrity was assessed by gel electrophoresis as described by BioRad Technical Note 5396 [18] using 500 ng of the extracted RNA and evaluation of the ratio between the bands corresponding to 16S and 23S ribosomal RNA. Reverse transcription of 1 μg extracted RNA in 20 μl reactions was performed with QuantiTect Reverse Transcription kit (QIAGEN) using random primers according to the manufacturer’s instructions. A control with omitted reverse transcriptase was performed for each extraction to check for the presence of contaminating genomic DNA. After reverse transcription, the samples were diluted 1:10 in DEPC treated H2O and used as templates for qPCR.

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Manufacturer protocol

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