Extraction of nucleic acids

2.4.1 RNA extraction from intact cells with DNase treatment

Prior to RNA extraction, cells were collected, transferred to 1.7 ml tubes and pelleted by centrifugation. Total RNA was extracted from cells using the RNeasy Mini  Kit  (Qiagen,  Valencia,  CA,  USA),  according  to  the  manufacturer’s  protocol.  

Qiagen supplied all the reagents provided within the RNeasy Mini kit and the 70% (v/v) ethanol was purchased from Merck.

Cell samples (<5 x 106 cells) were lysed with 350 l of Buffer RLT. For low cell number samples, culture media was removed from culture flasks and cells washed twice with PBS and 350 l of lysis Buffer RLT added directly to the cells and gently agitated for up to 5 minutes to promote cell lysis before transfer to 1.7 ml tubes. Samples were lysed thoroughly by pulse vortexing and pipetting to produce a lysate. The lysate was homogenized using a QIAshredder spin column (Qiagen). The lysate was transferred into a QIAshredder spin column assembled in a 2 ml collection tube, centrifuged at 13,000 rpm for 2 minutes and the flow-through retained. 350 l of 70% (v/v) ethanol was added and mixed gently by pipetting and inverting the tubes. The lysate/ethanol solution was applied to an RNeasy spin column assembled in a 2 ml collection tube and centrifuged at 13,000 rpm for 1 minute. The flow-through was discarded and the collection tube reused in subsequent steps. 350 l of Buffer RW1 was added to the spin column and centrifuged at 13,000 rpm for 1 minute and the flow-through discarded.

To remove any contaminating genomic DNA, samples were treated with DNase I. 80 l of DNase I (30 Units; Qiagen) in Buffer RDD was applied to the spin column and incubated at room temperature for 30 minutes. Following incubation, 350 l of Buffer RW1 was added to the spin column and centrifuged at 13,000 rpm for 1 minute and the flow-through discarded. 500 l of Buffer RPE was applied to the spin column and centrifuged at 13,000 rpm for 1 minute and the

flow-through discarded. A second application of 500 l of Buffer RPE was added to each spin column and centrifuged at 13,000 rpm for 2 minutes and the flow- through discarded. The spin column was transferred to a clean 2 ml collection tube and centrifuged for an additional minute at 13,000 rpm to ensure all residual wash solutions had been removed. The spin column was placed into a clean 1.7 ml tube and RNA recovered by elution with the addition of 50 l of RNase-free water directly to the spin column membrane and centrifuged at 13,000 rpm for 1 minute. Total RNA was stored at -80oC until required.

2.4.2 RNA extraction from tumor samples

Up to 30 g of tumor tissue was isolated using a clean scalpel blade and transferred to a clean 5 ml tube. 700 l of lysis Buffer RTL was applied and tumor samples were disrupted using a Pro 200 tissue homogenizer (Pro Scientific, Oxford, USA). 700 l of 70% (v/v) ethanol was added to the sample and mixed by pulse vortexing. Up to 700 l solution was applied to an RNeasy spin column assembled in a 2 ml collection tube and centrifuged at 13,000 rpm for 1 minute. This step was repeated until all of the solution had been applied to the RNeasy spin column. Total RNA was extracted from samples and DNase treated by following the protocol, as described in RNA extraction from intact cells

with DNase treatment (Section 2.4.1). Total RNA was stored at -80oC until

2.4.3 DNA extraction from intact cells

Total DNA was extracted using the DNeasy Blood and Tissue Kit (Qiagen), according   the   manufacturer’s   protocols.   Pelleted   cell   samples   were   resuspended in 200 l PBS and 20 l of Proteinase K (20 mg/ml; Qiagen) and 4

l RNase A (100 mg/ml; Qiagen) was added to remove any contaminating protein and RNA. 200 l of Lysis Buffer AL was added to each sample and mixed by pulse vortexing and incubated at 56oC for 15 minutes.

For direct cell lysis from culture plates, culture media was moved and cells washed twice with PBS. PBS was removed and the lysis solution of PBS, Proteinase K, RNase A and Buffer AL (total 424 l) were added directly to the cells and incubated at room temperature for 5 minutes. After incubation the lysis solution was gently triturated and transferred to a clean 1.7 ml tube. The samples were pulse vortexed for 15 seconds and incubated at 56oC for 15 minutes.

Following incubation, 200 l of 96-100% (v/v) ethanol was added to each sample and mixed thoroughly by pulse vortexing. The lysate/ethanol solution was transferred to a DNeasy Mini spin column assembled in a 2 ml collection tube and centrifuged at 13000 rpm for 1 minute, the flow-through was discarded and the spin column transferred to a new collection tube. 500 l of Buffer AW1 was applied to the spin column and centrifuged at 13000 rpm for 1 minute and the flow-through discarded and the spin column transferred to a clean collection tube. 500 l of Buffer AW2 was applied to the spin column and centrifuged at 13,000 rpm for 3 minutes to dry the spin column filter. The flowthrough was

discarded and the spin column was transferred to a clean collection tube and centrifuged at 13,000 rpm for 1 minute to remove any residual wash solutions. The spin column was placed into a clean microcentrifuge tube and 100-200 l of Elution Buffer AE applied. Spin columns were incubated at room temperature for 1 minute and centrifuged at 13000 rpm for 1 minute to recover DNA. DNA was stored at -20oC until required.

2.4.4 DNA extraction from tumor samples

Up to 30 g of tumor tissue was isolated using a clean scalpel blade and transferred to a clean 5 ml container. 180 l of Tissue Lysis Buffer ATL, 20 l Proteinase K (20 mg/ml) (Qiagen) and 4 l RNase A (100 mg/ml) was applied to the tissue sample. Tissue samples were disrupted using a tissue homogenizer and incubated overnight at 56oC. Following incubation, the solution was centrifuged at 13,000 rpm for 2 minutes and the supernatant carefully transferred to a clean 1.7 ml tube. 200 l of Buffer AL was applied to the solution, mixed by pulse vortexing and incubated for a further 10 minutes at 56oC. After incubation 200 l of 100% (v/v) ethanol was added to the solution and mixed by pulse vortexing. The solution was applied to a DNeasy Mini spin column assembled in a 2 ml collection tube and samples were further processed, as described in DNA extraction from intact cells (Section 2.4.3). DNA was stored at -20oC until required.

2.4.5 Quantification of nucleic acids

Extracted RNA and DNA was quantified and assessed for purity using the NanoDrop ND-1000 UV-Vis Spectrometer. The NanoDrop loading surface was cleaned and 1.5 l of dH2O applied to initialize the ND-1000 Software Version

3.2.0 (NanoDrop Technologies, Wilmington, USA). A second application of 1.5

l dH2O was used to blank the ND-1000 software to register a zero value. 1.5 l

of RNA/DNA was applied to the NanoDrop and absorbance was measured at 220-350 nm. Ratios of absorbance at 260/280 nm and 260/230 nm were calculated by the ND-1000 Software. Ratios of 1.7-2.1 indicated good quality and a high level of purity of RNA/DNA.