Cell Viability

Cell viability for both CLL and B lymphoid cell lines was checked before and after the different treatments using trypan-blue exclusion. Using this method live and dead

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cells can be distinguished; dead cells with disrupted plasma membranes will take up the dark blue dye, while live cells will appear shiny bright. As with the procedure for cell counting, cell suspensions were diluted 1:10 with 0.1% trypan blue dye solution, and dead and live cells were counted using a haemocytometer. Both live and dead cells were counted, and results reported as percent live cells within the total cell count.

2.3

Molecular Biology Techniques

2.3.1Nucleotide Studies

2.3.1.1 Total RNA Extraction

Cells harvested from experiments were collected in 1.5 ml nuclease-free eppendorf tubes (Anachem Ltd, UK). Total RNA was isolated from these cells using a ZR RNA Midiprep™ kit (Zymo Research, UK) following the manufacturer’s instructions. In brief, the cell pellet was lysed with 400 μl RNA lysis buffer and then centrifuged at 12,000xg for 1 min. The supernatant was transferred to a Zymo-Spin IIIC column, and this was centrifuged for 30 s at 8000xg. The flow-through was mixed with 320 μl of 100% ethanol, and then transferred to a Zymo-Spin IIC column where it was centrifuged at 12000xg for 1 min. The column with attached RNA was washed once with 400 μl RNA prep buffer, once with 800 μl and then with 400 μl RNA wash buffer using 30 s pulses of centrifugation at 12000xg. An additional centrifugation step involved 2 mins at 12000xg in order to completely remove residual wash buffer. As a final step RNA was eluted from the column with 30 μl of elution buffer and centrifugation for 30 s at 10000xg. Isolated RNA was immediately stored at -80 ᴼC following assessment for purity and quantity.

2.3.1.2 Assessment of RNA purity and quantity

The assessment of isolated RNA quality and quantity is essential for further molecular biology work. Quality of the isolated RNA from section 2.3.1.1 was assessed using a Nandrop 2000 spectrophotometer (Thermo scientific, UK). The isolated RNA was considered to be pure if the ratio light absorbance at 260 nm and

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280 nm fell within the range 1.6-2; a value below 1.6 indicates extensive protein contamination. An additional ratio of light absorbance was taken, A260nm/A230nm and

values between 2-2.2 were taken a pure; values outside this range indicate possible peptide or other contamination.

Quantitation of RNA within the isolation preps was also performed with the Nanodrop 2000. To do this, the absorbance value at 260nm was taken and divided by 0.025 (the extinction coefficient for single stranded RNA) to obtain the concentration in ng/l.

2.3.1.3 Synthesis of complementary DNA (cDNA) from RNA

To synthesize cDNA from isolated RNA, 1g of RNA was mixed with 1 µl of Oligo (dT) primer [500 ng/µl (Eurofins MWG Operon, Ebensburg, Germany)], and this was topped up with the addition of nuclease-free distilled water to a final volume of 14 µl. This was incubated for 5 min at 37ᴼC, and afterwards chilled on ice. Meanwhile, the reverse transcription master mix was prepared; per reaction 5 µl 5xRT Buffer, 1 µl (10,000U) Moloney murine leukaemia virus reverse transcriptase, 1 µl (10 mM) dNTP mix, 1 µl (2,500 U) RNase plus Rnase inhibitor (all from Promega, Southampton, UK) and 3 µl nuclease free water were mixed. To start the cDNA synthesis 11 µl of master mix was added to the 14 l of RNA/oligo dT mix. Synthesis was complete following incubation of the mixture at 42ᴼC for 1 h. Synthesized cDNA was kept in -20ᴼC until further used.

2.3.1.4 Polymerase Chain Reaction (PCR)

The basic RT-PCR reaction contained 4 µL of Hot Fire pol EvaGreen qPCR master mix (newmarketScientific, Kennet, UK ), 1 µl DNA (cDNA (prepared in section 2.3.1.3) or chromatin (prepared in section 2.3.6), 1 µl each (5 pmol/µl) of the forward and reverse primers (Eurofins MWG Operon, Ebensburg, Germany) needed to amplify the gene of interest, and 13 µl nuclease-free water to make a final reaction of 20 l. Amplification was performed using a DNA Engine® RPTC-200 Peltier thermal cycler (MJ Research, Watertown, Massachusetts, USA), and the following thermal profile: An initial step of heating to 95ᴼC for 1 min. This was

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followed by 40 cycles of denaturation at 95ᴼC for 30 s, annealing for 30 s at a temperature specific for the primers being used (this information is tabulated for each primer pair in Appendix C), and extension at 72ᴼC for 30 s. A final step consisted of extension at 68ᴼC for 5 min. With each PCR reaction a control experiment was included where nuclease-free water was added in place of DNA. Each PCR reaction was verified to amplify a single product of the correct size by agarose gel electrophoresis.

2.3.1.5 Quantitative Polymerase Chain Reaction (qPCR)

Quantitative, or real time, polymerase chain reaction is a powerful molecular technique that allows either absolute or relative quantification of specific sequences of DNA. Generally, quantification of amplified sequences is achieved by measuring, in real time, the fluorescence signal that is released from a dye, such as SYBR green dye, as it binds to the minor groove of newly synthesised DNA double strands during the elongation phase of the PCR reaction. Relative quantification is performed using a reference gene, whereas absolute quantification is determined from a standard curve. This technique has been used in various applications such as gene expression analysis, gene amplification studies and chromatin immunoprecipitation (ChIP).

The basic PCR reaction in this study is basically the same as is listed in section 2.3.1.4. The exceptions are that amplification took place using a Stratagene MX3000P PCR machine (Agilent Technologies, Stockport, UK). The cycling conditions were slightly changed to include a fluorescence measurement step of 11 s following the elongation step, and following the last cycle a melting curve was generated consisting of a final heat cycle of heating to 95ᴼC for 1 min, cooling to 55ᴼC for 30 s and reheating to 95ᴼC for 30 s where fluorescence is measured. Generation of the melting curve assayed for purity and specificity of the amplified products by appearance of a narrow single peak.

All PCR reactions had the same optimized cycling conditions with the exception that the temperatures for primer annealing and where the fluorescence data was

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collected were different used. This information is listed for specific primer pairs and genes of interest in Appendix C.

RNA polymerase II (For: 5’-CAAGACTGCTGAGACTGGATAC-3') and (Rev: 5'- CAAAGCGGAACTTCTTCTCAAAAG-3') was run as a reference gene for expression analysis of targeted genes in this study. RNA polymerase II was chosen because it showed a constant Ct value in qRT-PCR for all samples and different experimental conditions. As well, it is reported that RNA polymerase II expression is the most robust and constant reference gene within a comparison of all the classical reference genes (β-actin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), TATA-Box binding protein (TBP), hypoxanthine–guanine phosphoribosyltransferase (HPRT), Peptidyl prolyl isomerase A (PPIA), glucose 6-phosphate dehydrogenase (G6PDH), ribosomal protein L13(L13), β2-microglobulin (β2M), phospholipase A2 (PLA), α-tubulin (Tub), albumin (Alb) and Porphobilinogen deaminase (PBGD); when measured in cells in different studies under diverse experimental conditions [247]. To normalize target gene expression to RPolII, the following equation was used: