Illumina HiSeq 2000 paired end sequencing; Small non-coding RNAs as biomarkers of musculoskeletal ageing and osteoarthritis
Small non-coding RNAs as biomarkers of musculoskeletal ageing and osteoarthritis
Serum collected from heart following sacrifice. old normal donor 100ng of small RNA was added in a volume of 6ul together with 1ul of 3'SR adapter (diluted 1+2). This was heated at 70 degrees for 2minutes and cooled on ice. Ligation buffer and enzyme were added and the reaction incubated for 1 hour at 25 degrees. 2ul of Tobacco Acid Pyrophosphatase (TAP)(Epicentre) 5u/ul was added to each reaction and incubated at 37 degrees for 1 hour. Tobacco Acid Pyrophosphatase (TAP) hydrolyzes the phosphoric acid anhydride bonds in the triphosphate bridge of the cap structure found in most eukaryotic mRNA, releasing the cap nucleoside and generating a 5 ́-monophosphorylated terminus on the RNA molecule. This is inactivated in the subsequent step which heats the samples to 75 degrees for 5 minutes The samples were hybridized to the Reverse Transcription Primer at a dilution of 1+2 from the supplied stock of primer. This step is important to prevent adaptor-dimer formation. The SR RT Primer hybridizes to the excess of the 3 ́ SR Adaptor (that remains free after the 3 ́ ligation reaction) and transforms the single stranded DNA adaptor into a double-stranded DNA molecule. dsDNAs are not substrates for ligation mediated by T4 RNA Ligase 1 and therefore do not ligate to the 5 ́ SR Adaptor in the subsequent ligation step. Following this 5”ligation for 1 hour at 25 degrees, a reverse transcription was carried out at 50 degrees for 1 hour using ProtoScript II Reverse Transcriptase. The samples were then amplified for 15 cycles and incorporating a barcode as part of the priming process.  The samples were mixed into 3 pools,(mixing across groups) and size selected on a Sage Pippin cassette 2% DF marker E using a size range 120-300bp. The size selected material was purified with Ampure beads(1:1) (Agencourt). The pools were checked by Qubit analysis and Bioanalyser High Sensitivity chip.
Experimental Factor: Old
developmental stage
Experimental Factor: none
disease
Experimental Factor: Serum
organism part
Experimental Factor: none
treatment