TOTAL RNA ISOLATION FROM STREPTOMYCES CULTURES BY TOTAL NUCLEIC ACID PRECIPITATION AND DNase DIGESTION

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1 TOTAL RNA ISOLATION FROM STREPTOMYCES CULTURES BY TOTAL NUCLEIC ACID PRECIPITATION AND DNase DIGESTION Vassilis Mersinias Materials 2xYT medium (for pregermination of spores) RNaseZap (Ambion Cat# 9780) Modified Kirby Mix (1g N-Lauroylsarcosine (sarkosyl, Sigma Cat# L-9150), 6g p-aminosalicylic acid sodium salt (Sigma Cat# A-3505), 5ml 2M Tris-HCl ph8, 6ml buffered phenol ph8, dh 2 O to 100ml) Phenol/Chloroform/Isoamyl alcohol 25:24:1 (Phenol buffered at ph8 with Tris) Chloroform Isopropanol 3M Sodium Acetate ph 5.2 (in RNase-free H 2 O) 70% Ethanol (in RNase-free H 2 O) RQ1 RNase-free DNase (Promega Cat# M6101) RNeasy Protect Bacteria Midi Kit (Qiagen Cat# 75552) OR RNeasy Midi Kit (Qiagen Cat# 75142) and RNAprotect Bacteria Reagent (Qiagen Cat# 76506) Sterile universal bottle with ~14g glass beads mm diam. 25ml beaker Blunt-ended spatula 15ml tubes RNase-free pipette tips RNase-free Eppendorf tubes RNase-free deionised H 2 O Equipment Sonicator UniS Streptomyces microarray group. Total Nucleic Acids protocol Aug 03, page 1 of 5

2 Refrigerated bench centrifuge at room temperature and at 4 0 C with adaptors for 15ml and/or 50ml tubes Method Culture preparation (plates) Pregerminate Streptomyces spores in 50ml 2xYT for 6-10 hours. Pellet the cells at 3,000rpm for 10min. Resuspend the pellet in 10-12ml sterile H 2 O and sonicate in a sonic bath for 10min to disrupt clumped mycelia. Read the optical density at 450nm. Calculate the concentration (C) and the volume (V) required to inoculate 3x10 6 pregerminated spores (gs): C = (OD 450 / 0.04) x 4 x 10 6 gs/ml = OD 450 x 10 8 gs/ml V = (3 x 10 6 / C) x 1000 ml Inoculate solid media on plates overlaid with cellophane disc, with V ml of pregerminated spores. (Preparation of plates overlaid with cellophane discs: Boil the cellophane membranes in dh 2 O at 2 cycles of 10min. Change the water between cycles. Drain, add fresh dh 2 O and autoclave. Use two pairs of sterile forceps to lay the membranes on the plates. Exclude any bubbles. Dry the plates in the laminar flow for a few minutes. Do not overdry as this will make the cellophane to shrink. Use the plates on the same day or store in a sealed plastic bag at 4 0 C.) For liquid cultures (e.g. SMM) follow the instructions in the Streptomyces laboratory manual, p. 52 (Kieser et al., 2000). Stabilisation of RNA After growth harvest the mycelia from the plates with the spatula and resuspend them in 5ml RNAprotect Bacteria Reagent in a universal bottle containing glass beads. Vortex for 1min and leave on the benchtop for 5min. Then transfer the frothy suspension into a 15ml tube with a 1000P pipette (cut the edge of the tip with a scalpel to facilitate the recovery of the mycelia). If necessary wash the beads with an additional 1ml of RNAprotect Bacteria Reagent to recover any remaining mycelium. Centrifuge for 10min at room temperature at max speed. Discard all UniS Streptomyces microarray group. Total Nucleic Acids protocol Aug 03, page 2 of 5

3 supernatant. Remove remaining supernatant by gently dabbing the inverted tube onto a paper towel. Proceed to cell lysis or store the pellet in the freezer as described in the Qiagen manual. In case of liquid culture add 1 volume of the culture into 2 volumes of RNAprotect Bacteria Reagent in a 15ml or 50ml tube and vortex for 5s. Leave the cell suspension at room temperature for 5min. Pellet the cells at max speed for 10min at room temperature. Discard all supernatant. Remove remaining supernatant by gently dabbing the inverted tube onto a paper towel. Proceed to cell lysis or store the pellet in the freezer as described in the Qiagen manual. Cell lysis Resuspend the pellet in 5ml modified Kirby Mix and transfer the suspension into the same universal bottle containing glass beads. Vortex for 2min. Transfer the suspension into a 25ml beaker by pipetting. Add another 1ml modified Kirby Mix into the universal to wash the remaining mycelia and collect it in the beaker. Place the beaker on ice and sonicate the suspension at full power for 6 cycles 30s ON / 20s OFF. You should be able to observe a homogenized frothy sonicate. Clean the probe with 70% EtOH before and after sonication. Extraction, purification and precipitation of Total Nucleic Acids (TNA) Pour the sonicate into a fresh 15ml tube, add 1vol Phenol/Chloroform/Isoamyl Alcohol and vortex for 30s. At this point you can store the sample at C. Centrifuge for 10min at 4 0 C at max speed. Transfer the clear supernatant into a fresh 15ml tube by pipetting. Add 1vol Phenol/Chloroform/Isoamyl Alcohol, vortex for 30s, centrifuge as before and transfer the clean supernatant into a fresh tube. Add 1vol chloroform, vortex for 30s and centrifuge as before. Transfer the clear supernatant into a fresh tube and add 1/10vol sodium acetate and 1vol isopropanol. Incubate at room temperature for 5min and centrifuge for 10min at 4 0 C at max speed. Discard the supernatant and add 1-2ml 70% ethanol to wash the TNA pellet. Centrifuge as before and discard all the supernatant. Use a pipette to remove any residual ethanol and leave the tube open in a laminar flow in a angle from the surface towards the air flow. Dry the pellet for 20-30min. UniS Streptomyces microarray group. Total Nucleic Acids protocol Aug 03, page 3 of 5

4 DNase digestion Dissolve the pellet in 800ml RNase-free H 2 O. Add 100ml RQ1 buffer 10x and 100ml (100 units) RQ1 RNase-free DNase (the quantity of DNase is sufficient to digest the DNA in the expected yields of TNA). Incubate at 37 0 C for at least 30min. RNA purification For RNA destined for cdna synthesis and labelling we recommend the use of RNeasy Midi columns rather than conventional phenol extraction for the purification after DNase treatment. The method is described in the manufacturer s manual (RNA cleanup) with an extra wash step with Buffer RW1 (see below). Due to the maximum capacity of the column (1mg) first estimate the concentration of RNA by spectrophotometry at 260nm. Based on this rough concentration take an aliquot containing up to 900mg of impure RNA and purify on the RNeasy column. Additional on-column DNase digestion is optional. Before the first Buffer RPE wash step, perform an extra wash with addition of 4ml Buffer RW1, incubation on the benchtop for 5min and centrifugation for 5min (in case of on-column DNase digestion RW1 wash is modified; check Appendix E in the manual). Elute the RNA with 250ml RNase-free water. For the second elution step pass the first eluate through the column. This will give the desired RNA concentration for cdna synthesis and labelling. Do not discard the column until you quantify your RNA prep. If the concentration is not high enough perform another elution with fresh RNase-free water, combine the two eluates, freeze-dry and resuspend in a small volume of RNase-free water. Spectrophotometry Quantify the RNA at 260nm and measure 260/280 ratio ( for a pure preparation). We recommend the use of autoclaved ddh 2 O rather than DEPC-treated H 2 O for the dilution of the sample prior to spectrophotometry. Agarose gel electrophoresis Decontaminate an electrophoresis tank, gel tray and comb with RNaseZap. Check the RNA on an Agilent BioAnalyzer or a 1% agarose gel containing 0.2% iodoacetatic acid sodium salt UniS Streptomyces microarray group. Total Nucleic Acids protocol Aug 03, page 4 of 5

5 (Sigma Cat# I2512) as RNase inhibitor and EtBr. Use 1x TAE running buffer. The 23S and 16S rrna bands should be present with an intensity ratio ca. 2:1. A third band of a 23S-16S rrna aggregate is possible above the 23S rrna band. No genomic DNA on the top or smear at the bottom (degraded RNA) should be present. Store RNA preparation at C (-20 0 C should be used only for temporary storage). General Notes 1. Decontaminate bench surface and pipettors with RNaseZap. 2. Change gloves regularly. 3. If working with plate cultures start with cells from 6-7 plates in the early log phase, 4-5 plates in the mid or late log phase, or 3-4 plates in the stationary phase (numbers taken from cultures grown on Oxoid Nutrient Agar). microarrays@surrey.ac.uk UniS Streptomyces microarray group. Total Nucleic Acids protocol Aug 03, page 5 of 5