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Reverse transcription real-time quantitative polymerase chain reaction (RT-qPCR)

2.6.1.1 cDNA synthesis

The traditional reverse transcription was performed using miScript II RT Kit (Qiagen). RNA was thawed on ice. 10× miScript Nucleics Mix, RNase-free water, and 5× miScript HiFlex Buffer were thawed at RT. The reverse-transcription reaction mix was prepared on ice according to Table 4. The reactions were

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incubated for 60 min at 37 °C following an incubation at 95 °C for 5 min. The reactions were then placed on ice to cool down. The cDNA was then diluted 10 times with RNase-free water and stored at -20 °C for later use.

Table 4. Reverse-transcription reaction components for cDNA preparation for RT- qPCR.

Components Volume

5x miScript HiFlex Buffer 2 μl 10x miScript Nucleics Mix 1 μl miScript Reverse Transcriptase Mix 1 μl

Template RNA 6 μl

Total volume 10 μl

2.6.1.2 RT-qPCR

RT-qPCR was performed with miScript SYBR® Green PCR Kit (Qiagen) and miScript Primer Assays (Qiagen). MiScript Primer Assays (Qiagen) were reconstituted in 550 μl RNase-free water. The RT-qPCR mix was prepared on ice according to Table 5.

Table 5. RT-qPCR reaction components.

Components Volume/reaction(384-well plate)

2x QuantiTect SYBR Green PCR Master Mix

5 μl

10x miScript Universal Primer 1 μl 10x miScript Primer Assay 1 μl

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Template cDNA 1 μl

Total volume 10 ul

Reactions were loaded in optical 384-well reaction plates and run the programme (Table 6) on The Applied Biosystems™ QuantStudio™ 7 Flex Real-Time PCR System. Water was loaded as negative control. PC3 miRNA sample was loaded in every plate as normaliser between plates. All reactions were run as triplicates.

Table 6. Cycling conditions for RT-qPCR.

Step Time Temperature

PCR initial activation step 15 min 95 °C 3-step cycling Denaturation 15 s 94 °C Annealing 30 s 55 °C Extension 30 s 70 °C

Cycle number 40 cycles

Fluidigm multiple RT-qPCR

Samples for Fluidigm multiple RT-qPCR were prepared using the miScript Microfluidics PreAMP Kit (Qiagen), miScript Microfluidics PCR Kit (Qiagen), and miScript Primer Assays (Qiagen).

2.6.2.1 cDNA synthesis

Reverse transcription was performed using miScript II RT Kit (Qiagen). RNA was thawed on ice. 10× miScript Nucleics Mix, RNase-free water, and 5× miScript

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HiSpec Buffer were thawed at RT. The reverse-transcription reaction mix was prepared on ice according to Table 7. The reactions were incubated for 60 min at 37 °C following another incubation at 95 °C for 5 min. The reactions were then placed on ice to cool down. The cDNA was then diluted 5 times with RNase-free water and store at -20 °C for later use.

Table 7. Reverse-transcription reaction components for cDNA preparation for Fluidigm.

Components Volume

5× miScript HiSpec Buffer 2 μl 10× miScript Nucleics Mix 1 μl miScript Reverse Transcriptase Mix 1 μl

Template RNA 6 μl

Total volume 10 μl

2.6.2.2 Pre-amplification

The pre-amplification was performed using miScript Microfluidics PreAMP Kit (Qiagen) and miScript Primer Assays (Qiagen). MiScript Primer Assays (Qiagen) were reconstituted with 27.5 μl RNase free water to make a concentration of 100 μM. List of primer assays is shown in Table 8. To make miScript PreAMP Primer Mix, 2.2 μl of each primer assay was taken and mixed together with 1030 μl of RNase free water. The pre-amplification reactions were prepared according to Table 9.

Table 8. List of primer assays used in Fluidigm multiple RT-qPCR. MS00031829 - Hs_miR-375_2 miScript Primer

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MS00003640 - Hs_miR-184_1 miScript Primer MS00010752 - Hs_miR-9_1 miScript Primer MS00031703 - Hs_miR-320b_2 miScript Primer MS00003738 - Hs_miR-200a_1 miScript Primer MS00008372 - Hs_miR-101_3 miScript Primer MS00003556 - Hs_miR-148a_1 miScript Primer MS00003577 - Hs_miR-150_1 miScript Primer MS00032158 - Hs_miR-99a_2 miScript Primer MS00004179 - Hs_miR-423_1 miScript Primer MS00014707 - Hs_miR-320a_1 miScript Primer MS00004242 - Hs_miR-451_1 miScript Primer MS00003416 - Hs_miR-122a_1 miScript Primer MS00031234 - Hs_miR-100_2 miScript Primer MS00031710 - Hs_miR-320d_2 miScript Primer MS00009366 - Hs_miR-30c_2 miScript Primer MS00006552 - Hs_miR-24_1 miScript Primer MS00041867 - Hs_miR-320c_3 miScript Primer MS00048510 - Hs_miR-7706_1 miScript Primer MS00043491 - Hs_miR-1246_2 miScript Primer MS00048538 - Hs_miR-4433b-3p_1 miScript MS00009142 - Hs_miR-22*_1 miScript Primer MS00003535 - Hs_miR-146a_1 miScript Primer MS00003206 - Hs_miR-20b_1 miScript Primer MS00010549 - Hs_miR-744_1 miScript Primer MS00006629 - Hs_miR-125b_1 miScript Primer MS00008554 - Hs_miR-125a-3p_1 miScript

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MS00008281 - Hs_let-7b*_1 miScript Primer MS00009408 - Hs_miR-30e*_1 miScript Primer MS00007350 - Hs_miR-30a-5p_1 miScript Primer MS00003129 - Hs_let-7c_1 miScript Primer

Table 9. Pre-amplification reaction components for Fluidigm sample preparation.

Component Volume per

sample

5× miScript PreAMP Buffer 5 μl

HotStarTaq DNA Polymerase 2 μl

miScript PreAMP Primer Mix 5 μl

RNase-free water 7 μl

miScript Microfluidics Universal Primer 1 μl

Diluted cDNA 5 μl

Total volume 25 μl

Pre-amplification was then performed in thermal cycler according to Table 10.

Table 10. Cycling conditions for pre-amplification for Fluidigm sample preparation.

Step Time Temperature

PCR initial activation step 15 min 95 °C 2-step cycling:

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Annealing/extension 3 min 60 °C

Cycle number 12 cycles

After the run was finished, 2 μl Side Reaction Reducer was added to each pre- amplified reaction. The samples were mixed well, and centrifuged briefly to collect any liquid on the tube wall.

2.6.2.3 Multiple RT-qPCR

To perform multiple RT-qPCR, primers were further diluted from 100 μM to 40 μM by adding 7.5 μl RNase free water to 5 μl primer (100 μM). Pre-amplified cDNA samples were used without dilution. Multiple RT-qPCR was performed by Barts and the London Genome centre using BioMark HD system (Fluidigm, USA). Reactions were assayed in 96.96 Dynamic Array Integrated fluidic circuit. PCR was performed with 40 cycles.

RNA-sequencing

2.6.3.1 Exosome RNA-sequencing

Exosome RNA from 24 treatment-naive PCa and 24 CRPC samples were sent for RNA-sequencing to the Barts and the London Genome Centre core facility. Indexed libraries were prepared from exosomal RNA as instructed by the NEBNext Multiplex Small RNA Library Prep Set for Illumina (New England Biolabs) according to the manufacturer’s instructions without size selection. RNA- sequencing was performed on Illumina NextSeq 500 platform and run twice.

2.6.3.2 RNA-sequencing data analysis

Unaligned reads were trimmed and aligned against mirBase mature miRNA (Release 20) within Partek® Genomics Suite®. Low expression miRNAs (expressed in less than 25% of samples) were removed. The miRNA read counts were then analysed using three methods to identify differentially expressed

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miRNAs. 1) Linear Models for Microarray and RNA-sequencing Data (Limma) [340] was performed using functions implemented in edgeR and limma() in R studio. 2) Counts per million (CPM): Read counts were transformed to CPM using cpm() function implemented in R package edgeR (version 2.4.0) and then Welch’s t-test were performed in SPSS 24. 3) The trimmed mean of M-value normalisation (TMM)[341] was performed using functions implemented in the edgeR in R studio and ExactTest() function in edgeR was used to find differentially expressed miRNAs.