MiScript MiRNA PCR Array

Miscript miRNA PCR Array Brain Cancer panels (Qiagen, Manchester, UK), were used to select miRNA for a preliminary panel. Following isolation from serum samples, total RNA was reverse transcribed using the miScript RT II kit (Qiagen, Manchester, UK) and the expression level of 84 miRNAs associated with brain cancer were determined using qPCR. The C.elegans miR-39 miScript Primer Assay (Qiagen, Manchester, UK) was used as a control to normalise qPCR data.

Total RNA was isolated from serum and cerebrospinal fluid as outlined in Section 2.3.3 and from tissue as outlined in Section 2.3.4. The C.elegans miR-39 miScript Primer Assay) was diluted to a final concentration of 1.6 x108 copies/µl (Table 2.3) and added to the sample following homogenisation and lysis. Total RNA was diluted to a final concentration of 12.5 ng/µl and then reverse transcribed. The master mix components excluding the reverse transcriptase mix were thawed at room temperature and the master mix was prepared following Table 2.4. The reverse transcriptase was removed from -20 ˚C immediately before adding to the master mix and then immediately replaced into -20 ˚C to maintain integrity of the enzyme. The master mix was mixed by pipetting, centrifuged at 2000 x g for five seconds in a Technico mini centrifuge (Fisher Scientific, Loughborough, UK) stored on ice and then incubated at 37 ˚C for 60 minutes followed by 95 ˚C for five minutes.

Table 2.3: Dilution and Final Concentration of C. elegans miR-39 miScript Primer Assay.

Dilution Concentration (Copies/µl) Initial Stock 300 µl DEPC treated H2O + 10

pmol lyophilised sample

2 x 1010

Dilution 4 µl stock + 16 µl RNase-free water

4 x 109

51 free water

Table 2.4: Reverse Transcription Master Mix, Components and Final Working Concentrations.

Component Volume Final Concentration

5 X miScript HiSpec Buffer 4 µl 1 X

10 X miScript Nucleics Mix 2 µl 1 X

RNase-free water Variable -

MiScript Reverse Transcriptase Mix

2 µl -

Template RNA Variable 12.5 ng/μl

Total Volume 20 µl -

Following reverse transcription cDNA was diluted 1:10 in RNase free DEPC water (Life Technologies, Paisley, UK) prior to qPCR. The qPCR master mix components were thawed at room temperature and pipetted following Table 2.5. The brain cancer panels were thawed at room temperature, and 25 µl of reaction mix was added to each well and the plate sealed with optical adhesive film. The plate was then centrifuged at 1000 x g for one minute to ensure all the master mix was at the bottom of the well. The C.elegans, reverse transcription control and positive PCR control wells within the brain cancer panels were utilised in this study.

The qPCR reaction was performed on an Applied Biosystems 7500 Real Time PCR System in standard mode using the parameters outlined in Table 2.6 and a dissociation analysis step added at the end of the run.

Table 2.5: qPCR Master Mix and Final Working Concentration.

Component Volume (µl) Final Concentration

2 X QuantiTect SYBR Green PCR Master Mix

1375 1 X

10 X miScript Universal Primer 275 1 X

RNase free water 1000 -

Template cDNA (diluted) 100 -

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Table 2.6: qPCR Parameters.

The data were analysed using the miScript miRNA PCR Array Data Analysis tool (SABiosciences) available online at http://pcrdataanalysis.sabiosciences.com/mirna. Threshold cycle (Ct) values were normalised using the C.elegans miR-39 miScript Primer Assay (Qiagen, Manchester, UK). Normalisation was performed by subtracting the Ct value of the C.elegans spike in from the target miRNA Ct value. Each miRNA was scored either A, B or C by the miScript data analysis tool based on the average Ct to determine whether the calculated fold change was representative of the actual fold change within the sample (Table 2.7). ‘A’ being representative of the actual fold change and ‘B’ suggesting a variation in fold change between samples which may not be representative of the actual fold change. MiRNAs were scored ‘C’ if the average Ct of either the target sample or the control sample was beyond the defined cut off of 35 cycles, making the calculated fold change invalid. Following analysis using the miScript miRNA PCR Array Data Analysis tool, data was examined and miRNAs with a fold change scored as a ‘C’ were omitted from the study.

Table 2.7 Fold Change Scoring system defined by the miScript data analysis tool.

Score Definition

A This gene’s average threshold cycle is relatively high (> 30) in either the control or the test sample, and is reasonably low in the other sample (< 30).These data mean that the gene’s expression is relatively low in one sample and reasonably detected in the other sample suggesting that the actual fold-change value is at least as large as the calculated and reported fold-change result.

B This gene’s average threshold cycle is relatively high (> 30), meaning that its relative expression level is low, in both control and test samples.

Enzyme Activation Hold 15 minutes 95 ˚C

PCR - 40 Cycles Denature Hold 15 seconds 94 ˚C

Anneal Hold 30 seconds 55 ˚C

Extend Hold 34 seconds 70 ˚C

Melt curve analysis – 1

Cycle

53

C This gene’s average threshold cycle is either not determined or greater than the defined cut-off (default 35), in both samples meaning that its expression was undetected, making this fold-change result erroneous and un-interpretable.