Electron Scattering from Pyrimidine

2.8.1 Materials

Ribonucleic acid (RNA) extraction from cells was undertaken using a QIAshredder (cat.

79656) and RNeasy mini kit (cat. 74106). For RNA isolation from tissues, RNeasy Fibrous Tissue Mini Kit (cat. 74704) was used. RNase-Free DNase Set (cat. 79254) was used to eliminate genomic DNA contamination in the samples. Generation of cDNA was achieved using a QuantiTect® Reverse Transcriptase kit (cat. 205313), and a QuantiTect® SYBR®

Green PCR kit (cat. 204143) was used for qPCR reaction. All sourced from Qiagen (Hilden, Germany).

2.8.2 RNA extraction

2.8.2.1 RNA extraction from cardiac fibroblasts and cardiomyocytes

Total RNA extraction was performed according to the manufacturer’s instructions using a RNeasy mini kit (cat. 74106; Qiagen; Hilden, Germany). Briefly, cells were thawed on ice and pipetted directly into a QIAshredder spin column placed in a 2mL collection tube, and centrifuged for 2 minutes at 14000rpm. One equal volume of 70% ethanol was added to the homogenised lysate and mixed well by pipetting. The samples then transferred to an RNAeasy spin column placed in a 2mL collection tube and centrifuged for 15 seconds at 14000rpm. In order to eliminate genomic DNA contamination, on-column DNAse digestion was performed using a RNase-free DNase Set (Qiagen, Hilden, Germany): 350µL of buffer RW1 solution was added into the RNeasy spin column and centrifuged for 15 seconds at 14000rpm. 10µL DNase I solution plus 70µL of buffer RDD were added directly to the RNeasy spin column membrane and incubated for 15 minutes at room temperature. The columns were then washed with 350µL of buffer RW1 and centrifuged for 15 seconds at 14000rpm. After further washes with 500µL of buffer RPE twice, the RNeasy spin columns then placed onto new collection tubes, and centrifuged for 1 minute at 14000rpm to eliminate any possible carryover of RPE. The columns were then placed in 1.5mL tubes for RNA collection. 30µL of RNase-free water were used to elute the RNA on the spin column membrane (centrifuged for 1 minute at 14000rpm). The eluents were re-applied onto the spin columns and centrifuged to obtain a higher concentration of RNA.

2.8.2.2 RNA extraction from tissue

RNA extraction from tissues was achieved with a RNeasy Fibrous Tissue Mini Kit (Qiagen, Hilden Germany) and performed according to the manufacturer’s instruction. Organs were powdered using a pestle and mortar in liquid nitrogen. 25-30mg of tissue sample was dissociated in 300μL RLT buffer with 3μL β-mercaptoethanol (Sigma-Aldrich, Poole, UK) by agitation using a 1mL pipette. The cell suspension were then homogenised with a QIAshredder. 590μL of H2O and 10μL of proteinase K were added to the homogenised cells and incubated at 55°C for 10 minutes followed by 3 minutes centrifugation at 14000rpm.

The supernatant were transferred to 1.5mL tubes and 450uL of 100% ethanol was added and mix by pipetting. The mixture was then transferred to RNAeasy spin columns and spun at 14000rpm for 15 seconds for total RNA binding on the column membrane. The on-column genomic DNA elimination and RNA eluting steps were then performed as described above.

2.8.3 Measurement of RNA concentration and quality

The concentration and quality of RNA was determined using a Nano-drop®ND-1000 Spectrophotometer (Thermo-Fisher, Leicestershire, UK). Sample concentration in ng/uL was calculated based on absorbance at 260 nm. The ratio of sample absorbance at 260 and 280 nm (260/280) is used to assess the purity of RNA. A ratio of ~2.0 is generally accepted as “pure” for RNA. If the ratio is <1.8, it may indicate the presence of protein, phenol or other contaminants that absorb strongly at or near 280 nm, and the RNA sample was excluded. The ratio of sample absorbance at 260 and 230nm (260/230) is a secondary measure of nucleic acid purity. The 260/230 values for ‘pure’ nucleic acid are often higher than the respective 260/280 values and they are typically in the range of 1.8-2.2. If the ratio is appreciably lower, it may indicate the presence of ethanol or guanidine contamination (NanoDrop 1000 Spectrophotometer V3.8 User's Manual).

2.8.4 Complementary (c)DNA generation

Since the yield of total RNA extracted from neonatal cardiomyocytes was relatively low (approximately 40ng/µL), 250ng of RNA were used to generate cDNA. Whereas, the yields from cardiac fibroblast (approximately 200ng/µL) and tissues (>400ng/μL) were relatively high and thus, 1000ng of RNA were used to generate cDNA. First, genomic DNA elimination step was carried out by adding 2µL of gDNA wipeout buffer (Qiagen; Hilden, Germany) to 12μL of RNA samples and followed by an incubation of 2 minutes at 42°C. A reverse transcription (RT) master mix prepared according to Table 4 was then added to the

samples. A thermal cycler (Bio-Rad S1000^TM, UK) was used for the reverse transcription reaction (15 minutes at 42°C followed by 3 minutes at 95°C to inactivate Quantiscript Reverse Transcriptase). The cDNA products were stored at -20°C until proceeding to real-time quantitative (q)PCR.

2.8.5 Real-time quantitative PCR 2.8.5.1 cDNA dilution

Prior to the qPCR reaction, cDNA was diluted 1:40 with RNase-free water, except for the detection of the Nppc gene, in which cDNA was diluted 1:2 due to low mRNA expression.

2.8.5.2 qPCR reactions

The components for the qPCR master mix per reaction were prepared according to Table 5, and samples were prepared in triplicate. The qPCR reactions were facilitated by the 7900HT machine and the qPCR cycle conditions for 10μL sample are shown in Table 6. Using the SDS 2.4 software, the efficiency of the qPCR reaction was assessed by examining the melt curves for each reaction to exclude primer-dimer formation and to ensure that only one product was amplified. The threshold (manual Ct) for each detector was adjusted by placing the threshold line at the geometric phase along the amplification plot. The baseline for each detector was set manually to end before the amplification curve starts to rise.

Details of the primers used are shown in Table 7. β-actin and RPL-19 were used as reference genes and relative gene expression were calculated using the 2^-ΔΔCt method (Livak and Schmittgen, 2001).

Master mix components for reverse transcription

Table 4. Master mix components for reverse transcription per reaction.

Master mix components for qPCR reaction

Table 5. Master mix components for qPCR reaction per sample.

Real-time qPCR cycle conditions

Table 6. qPCR cycle conditions.

Cardiac remodelling target genes and their primer sequence

Table 7. List of cardiac remodelling target genes and their primer sequences for qPCR.