MiRNA Isolation and Quantification

2.2.1 MiRNA Extraction from plasma samples

Extraction of miRNA was performed using the miRNeasy Mini Kit (Qiagen, Hilden, Germany Cat. No. 217004) which is designed for purification of total RNA, including miRNA and other small molecules, from cultured cells and both human and animal tissues. This kit combines phenol/guanidine-based lysis of samples and the silica membrane-based purification of total RNA.

2.2.1.1 Extraction

All work was done under a fume hood and on top of ice. 300 µL of plasma sample was separated into two 1.5 mL Eppendorf tubes (150 µL each tube). Five volumes (750 µL) of QIAzol Lysis Reagent (Qiagen, Hilden, Germany Cat. No.79306) was added to each tube and mixed by vortex until all precipitate disappeared. Tubes were then placed in the fume hood at room temperature for 5 minutes to ensure complete dissociation of nucleoprotein complexes. Chloroform (150 µL) was added next to all tubes containing the homogenate, mixed by vortex and allowed to stand for 3 minutes at room temperature. Following centrifugation for 15 minutes (8000 G, ͶԨ), the upper clear aqueous phase was separated into two new 1.5 mL Eppendorf tubes (250 µL each), making a total of 4 tubes for each plasma sample. 1.5 volume of 100% ethanol was added to each tube. One RNeasy Mini spin column was prepared for each sample by placing it into 2 mL collecting tube. All four tubes of the same sample were added to the same column. One by one, the samples were mixed by pipetting, collected (up to 700 µL) into the column, and centrifuged (8000 G, 4 minutes, at room temperature), with the flow-through being discarded. This was repeated three more times for the remainder of the samples. Buffers in the kit were prepared as instructed by the manufacturer. RNeasy Mini spin column was washed with 700 µL of RWT buffer and centrifuged (8000 G, 2 minutes, at room temperature). Flow-through was discarded. The column was washed again with 700 µL RPE buffer and centrifuged (8000 G, 2 minutes, at room temperature). Flow-through was discarded. 500 µL of 75% ethanol was added onto the RNeasy Mini spin column and centrifuged (8000 G, 2 minutes, at room temperature). The spin column was put into a new 2 mL collection tube and dried by centrifugation (8000 G, 5 minutes, at room

temperature). After that, the spin column was transferred onto a new 1.5 mL collection tube. 20 µL of RNase-free water was pipetted directly onto the spin column and centrifuged (8000 G, 2 minutes, at room temperature). Using the same flow-through, the previous step was repeated twice more to concentrate the RNA. The sample was kept on ice until nanodrop RNA quantification.

2.2.2 MiRNA extraction from Adipose tissue

MiRNAs were extracted from adipose tissue using the QIAGEN RNeasy Lipid tissue mini kit (QIAGEN, Crawley, UK, Cat. No. 74804).

2.2.2.1 Homogenization of Adipose Tissue Using the Tissuelyser

Adipose tissues were homogenized using the Tissuelyser (QIAGEN, Germany, Cat.No.85220). Approximately, 100 mg of adipose tissue was disruptive by rapid agitation in the presence of 5 mm mean diameter Stainless steel beads (QIAGEN, Cat.No.69989) and lysing buffer (1 mL of QIAzol).

2.2.2.2 Extraction

Following homogenization, 200 µL of chloroform was added. Mixture mixed thoroughly by vortex and samples spun at 12,000 g for 15 minutes at 4Ԩ. The aqueous layer (approximately 600 µL) was removed and placed in a fresh RNAse free tube. RNA was precipitated by addition of 70% ethanol and transferred to a RNeasy mini spin column. If necessary, samples were treated with DNase (See section DNase Treatment of RNA). Columns then underwent a series of washes to remove impurities before being eluted in 50 µL of RNase free H2O. RNA was stored at -80Ԩ.

2.2.2.3 DNase Treatment of RNA

Further DNA removal was necessary before analysis of miRNA by TaqMan qRT-PCR to remove DNA contaminants that can be detected during qRT-PCR and potentially cause experimental artefacts. DNase treatment was performed by incubation of samples in on-column DNase digestion set (QIAGEN, Cat.No.79254) according to manufacturer’s protocol. For each sample 10μl of DNase I stock solution was mixed with 70μl Buffer RDD, applied to column and allowed to incubate at room

temperature for 15 minutes. This step was performed during the first column wash of the miRNEasy protocol. Following DNAse treatment, the miRNeasy wash protocol was continued.

2.2.3 Total RNA Quantification using Nanodrop

The concentration of RNA in each sample was measured using the Nanodrop 1000, micro-volume ultraviolet-visible spectrophotometer (Thermo Fisher Scientific, Waltham, Massachusetts v3.7) with the ND-100 software per the manufacturer’s instructions. 2 µL of nuclease free water was carefully pipetted onto the end of a fibre optic cable/receiving fibre and used as a blank. Subsequently, 2 µL of each sample was measured in a similar method to the water blank. Concentration measurements were recorded, along with the ratio of sample absorbance (260/280, 260/230) to check for nucleic acid purity as well as protein and phenol contamination. Measurements were taken twice and stored atെͺͲԨ.

2.2.4 MiRNA Reverse Transcription

MiRNA reverse transcription (RT) was done to generate complementary DNA (cDNA) from an RNA using the enzyme Reverse transcriptase. Taqman MicroRNA Reverse Transcription Kit was used (Applied Biosystems, Foster City, California, no.4366596) as per manufacturer’s protocol. Primers for each miRNA (RT, 5X) were used to prepare master mix.

Samples Preparation

A total of 9 ng miRNAs is required. The dilution was made using nuclease free water (Appendix 1). A fixed volume of 5 µL from the total RNA was used for the RT process.

Master Mix Preparation

One master mix was prepared for each miRNA primer. Each master mix contained dNTP (100 mM), multiscribe, 10X RT buffer, RNase inhibitor, nuclease free water and the specific miRNA primers allocated (5X, RT) (Appendix 1). The master mix was mixed and kept on ice.

Protocol

A 96-well plate was prepared, marked and kept on ice. 4.5 µL of diluted RNA samples were added to each well as allocated and the plate was spun. 3 µL of the master mix was added to each well and the plate was spun. The plate was sealed with adhesive sealing sheets and ran on a Polymerase Chain Reaction (PCR) block for RT reaction. Thermal cycle conditions were: _{ͳ͸Ԩfor 30 mins, ͶʹԨ for 30 mins,} ͺͷԨ for 5 mins, ͶԨ for 30 mins and then held at ͳʹԨ. The plate was kept in െʹͲԨ until used for QPCR.

2.2.5 Quantitative Real-Time Polymerase Chain Reaction (qRT-

PCR)

Quantitative real-time polymerase chain reaction (qRT-PCR) was performed using the 2X Universal TaqMan Master Mix (Applied Biosystems, Foster City, California) and TaqMan primers for each miRNA (TM, 20X). Samples were running in duplicate including the positive and negative QC.

Plate Preparation

The 96-well cDNA plate was taken out from െʹͲԨ and kept on ice until thaw. 7.5 µL RNase free water was added into each well and spun at 4Ԩto bring everything down.

Master Mix Preparation

Calculations of the amount of master mix required is shown in (Appendix 2). One tube was prepared for each miRNA primer. To each tube, 2X TaqMan master mix, 20X primers/probes, and RNase free water was added.

Protocol

An MicroAmp optical 384-well reaction plate was prepared, marked, and kept on ice.1.4 µL of the cDNA product was pipetted on the side of the well and centrifuged. After that, 8.6 µL of master mix was pipetted into each well, centrifuged and sealed with an optical adhesive lid.

Quantitative real-time PCR (qRT-PCR) was performed using the QuantStudio 7 Flex Real-Time PCR System (Applied Biosystems, Foster City, California V1.3). The Thermal cycling conditions were 95Ԩfor 10 mins., followed by 40 cycles of 95Ԩ for 15 s and 60_{Ԩ for 60 s. For each miRNA, four runs of qRT-PCR were done} including quality control (QC) sample and negative control sample (both in duplicate). Data were analysed with the QuantStudio 7 software (Applied Biosystems, Foster City, California V1.3) to determine the threshold cycle (Ct).