RNA TECHNIQUES

RNA was utilised to compare gene expression differences between samples based on time and the presence/absence of mercuric chloride in the media. Extracted RNA was used to produce cDNA, which was used for Reverse-Transcriptase PCR and Real-Time PCR analysis. The following sections detail these methods.

140

2.12 Isolation of RNA from bacterial cells

Prior to performing any RNA extractions, it was necessary to inactivate all RNAses from solutions and glassware. All solutions and water required for RNA work were first treated with diethyl pyrocarbonate (DEPC), as per manufacturer’s instructions. 100 μL of DEPC was added per 1000 mL of solution or water. The solutions were left at room temperature overnight in the fume hood, followed by autoclaving at 121oC for 15 minutes. Tris buffer solutions were prepared with DEPC-treated dH2O followed by autoclaving. All glassware to be used for RNA extractions was baked at 240oC for 24 hours.

2.12.1 Determination of fixed cell numbers for RNA extractions

RNA was extracted from the cells collected during the growth curve/induction experiments (Section 2.3.6), to study the expression of the mer genes at various time points and in the presence or absence of mercuric chloride. RNA was extracted from equal cell numbers (approximately 1.75 X 108) as described by Emslie (2002). To do this, the cell numbers from each sample were determined (Section 2.3.7). The cell pellet that was determined to contain the lowest cell number was resuspended in 10 mL protoplasting buffer. To account for differences in cell numbers within the pellets, the amount of protoplasting buffer added to resuspend each additional pellet was increased 10 ml of the resuspended volumes (corresponding in cell numbers to the cell pellet containing the lowest number of cells) was used for RNA extraction.

2.12.2 Isolation of RNA from Gram-negative bacteria

The method was based on that described by Ausubel et al., (1995). The cell pellet was resuspended in an appropriate volume of protoplasting buffer, 80 μL of lysozyme solution (50 mg/mL) was added to it, followed by incubation on ice for 15 minutes. The protoplasts were centrifuged at 5900 rpm for 5 minutes at 4oC, then resuspended in 0.5 mL Gram-negative lysing buffer and 15 μL DEPC-treated dH2O was added. The solution was mixed and incubated at 37oC for 5 minutes, then cooled on ice followed by the addition of 250 μL saturated NaCl. The solution was mixed and incubated on ice for 10 minutes followed by centrifugation at 14000 rpm for 10 minutes at 4oC. The

supernatant was removed to two new microfuge tubes and 1mL ice-cold 100% ethanol was added to each. RNA was precipitated at -80oC for 30 minutes and pelleted by centrifuging at 14000 rpm for 15 minutes at 4oC. The RNA pellets were rinsed in 500 μL of ice-cold 70% ethanol, air-dried at room temperature for 10 minutes and resuspended in 100 μL of DEPC-treated dH2O. To remove any residual DNA, the samples were later DNAseI treated (see below).

2.13 Quantitation of RNA samples

Extracted RNA samples were diluted and their absorbance taken as described for DNA quantification (Section 2.6). To determine the RNA concentration the following formula was used:

RNA: Absorbance reading X 200 (dilution factor) X 40 = μg/mL RNA. All RNA samples were electrophoresed as described in Section 2.7.

2.14 Reverse Transcription-PCR analysis

The following methods describe the DNase treatment of RNA samples, the synthesis of cDNA from the RNA and the amplification of the gene of interest from the cDNA.

2.14.1 Removal of residual DNA from RNA samples

RNA samples (prepared in Section 2.12.2) were DNase I treated to remove any residual DNA, by using the method based on that provided by New England Biolabs. Reactions were set up by adding: 5 μL (generally up to 10 μg) RNA, 1 μL DNase I (10U/µL) 1 μL RNasin, (2 0U/ μL) 1 μL 10X DNase b uffer an d 2 μL DEPC-treated water. These reactions were incubated at 37oC for 30 minutes, then stopped by adding 1 μL of 25 mM EDTA (pH 8.0) and incubating at 65oC for 15 minutes.

2.14.2 cDNA synthesis

The details of this method were provided with the SuperscriptII reverse transcriptase enzyme (Invitrogen). To produce cDNA, 1 μL of antisense strand primer (i.e., merR2,

142 merT2, merP2, mer32) or random primer (10 µM, 6 bases; Promega) and 5 μL of DEPC-treated dH2O was ad ded to 5 μL of DNase-treated RNA and the mixture was incubated at 70oC for 15 minutes. The sample was then split into duplicates (5.5 μL per tube) and 0.5 μL RNasin, 4 μL 5X first strand buffer,2 μL 0.1 M DTT and 1 μL 10 mM dNTP mix was added to each tube. These were incubated at 42oC for 2 minutes. 1 μL of Superscript II RTwas added to one of the duplicate tubes and both tubes incubated at 42oC for 50 minutes, followed by 70oC for 15 minutes. The tube containing no Superscript II enzyme serves as a negative control, to determine whether any amplification products in the RT-PCR were as a result of any residual DNA in the RNA sample after DNaseI treatment.

2.14.3 Reverse Transcription-PCR

PCR amplifications were set up using 2 μL of the above cDNA reaction mix, 5 μL 10X buffer, 2 μL 50 mM MgCl2, 1 μL 1 μL 10 mM dNTP mix, 3.5 μL (0.1 μg/μL) of each

forward and reverse primer for the gene to be amplified (Table 2.2), 25.5 μL water and 0.5 μL Taqpolymerase (5 U/ μL). The samples were then amplified as described in

Section 2.8.2. 5 μL of each PCR mix was electrophoresed to determine the presence of specific gene transcripts in the RNA samples (Section 2.7).

Chapter 3

Establishing the Heavy Metal Resistance of Achromobacter sp.