ACTIVIDAD # 14 Título: “ Creo mi texto ”

Total ribonucleic acid was isolated from PPP samples using the miRNeasy Mini kit (Qiagen, Hilden, Germany), using phenol- and chloroform-based extraction. This previously reported RNA extraction method is discussed in detail in this section.

The 174 samples were extracted sequentially in batches of 24. Following defrosting, 500µl of PPP were transferred to 1.5 ml Eppendorf tubes and centrifuged at 4,000rpm at 4oC for 10 minutes. To minimise RNA degradation, samples were kept on ice throughout.

An exogenous miRNA (spike-in) was added at the start of the extraction process to allow for normalisation of the RNA isolation. This Spike-in is a synthetic miRNA based on the sequence of cel-miR-39-3p in the C. Elegans nematode. This miRNA does not occur in mammals, allowing for its use as an exogenous control. Following reconstitution in RNase-

165 free H2O at a concentration of 10 µM, cel-miR-39-3p was further diluted to 2.5 nM with

RNAse-free H20 in two steps, being vortexed at each stage. A mixture was prepared to be

added to the sample, combining 4 µl of diluted cel-miR-39-3p, 194.75 µl of QIAzol lysis buffer, and 1.25 µl of bacteriophage-based carrier RNA (MS2). This mixture was prepared at the start of each sample batch, allowing for a sufficient amount for that batch.

QIAzol lysis reagent is a monophasic solution of phenol and guanidine thiocyanate, designed to denature protein complexes and RNases, alongside removing most of the residual DNA and protein from the lysate by organic extraction. Under a laminar flow fume hood, 500µl QIAzol reagent was placed in each Eppendorf tube and subsequently 100µl of each samples was added, which was then vortexed, 30x inverted and incubated at room temperature for 5 minutes. Subsequently 200µl Spike/QIAzol/MS2 mixture was added to each tube, followed by being immediately vortexed, 30x inverted and incubated at room temperature for 5 minutes.

Next 140µl chloroform was added to each Eppendorf tube, followed by being vortexed, shaken for 30 seconds and incubated at room temperate for 5 minutes. The Phenol to Chloroform ratio 5:1 is established with these additions, which is the optimal condition for producing conformational changes to proteins and lipids. Chloroform addition to phenol is more efficient at denaturing proteins than either reagent is individually. Furthermore, the addition of chloroform forces a sharper separation of the organic and aqueous phase during subsequent centrifugation as it is miscible with phenol and has a higher density than phenol (1.47g/cm3 vs 1.07g/cm3), which assists the removal of the aqueous phase with minimal

166 impact on the organic phase. Consequently, phenol on its own would retain 10-15% of the aqueous phase and result in a lower yield of RNA.

The pH of phenol determines the separation of RNA and DNA between the two phases. When the pH is neutral or minimally alkaline (pH7-8), the phosphate diesters in the nucleic acid are negatively charged, which results in retention of RNA and DNA in the aqueous phase. DNA is removed from the aqueous phase as the pH lowers, to a maximum effect at a pH 4.8. During transfer of DNA to the aqueous phase it dissolves, due to the negative charge in their phosphate groups being neutralised in acid by protonation. RNA despite being negatively charged remains in the aqueous phase, due to being single stranded and having exposed nitrogen bases, which allows it to form covalent bonds with hydrogen in H2O.

Altogether, acidic phenol causes retention of RNA in the aqueous phase and DNA in the organic phase; thus separating DNA from RNA. Moreover, during the centrifugation process and the subsequent separation, proteins contained within the samples separate out if they have charged domains or hydrophobic regions. These hydrophobic cores interact with the phenol causing precipitation at the interface between the two phases. The lipids in the sample dissolve in the lower organic phase.

Following incubation, the tubes containing sample, Spike/MS2 mixture, QIAzol and chloroform were centrifuged at 12,000rpm at 4oC for 15 minutes to cause phase separation. Following centrifugation, 280µl of upper phase were transferred to new Eppendorf tubes containing 480µl 100% ethanol, mixing together with a pipette. This step allows for RNA recovery by precipitating and separating it from contaminants, alongside providing

167 appropriate binding conditions for all RNA molecules from 18 nucelotide and above. After mixing thoroughly, the sample/ethanol mixture were added to the miRNeasy Mini spin column, which is then centrifuged at 13,000rpm for 1 minute at room temperature. Total RNA binds to the silica-membrane, a process enhanced by guanidinium. The flow-through was discarded. The next step involved washing out the phenol and remaining additional contaminants, which was performed by adding 700µl RWT buffer (buffer contents are considered proprietary information by the company and are therefore not disclosed) to the column. The columns were then centrifuged at 13,000 rpm for 1 minute at room temperature, with the flow-through being discarded. After this 500µl RPE buffer was pipetted into the column, and was centrifuged at 13,000 rpm for 1 minute at room temperature, with the follow-through being discarded. This step was repeated and followed by centrifugation for 2 minutes instead. The column was then placed in a clean collection tube and centrifuged at 15,000rpm for 1 minute at room temperature to facilitate further drying of the membrane.

The column was subsequently placed in a new Eppendorf tube and 35µl RNase-free H20 was

pipetted onto the membrane and centrifuged at 9,500 rpm for 1 minute at room temperature. This process elutes the RNA into the H20. The tube containing the RNA were

then stored at -80 oC until further use.