The overall goal of this procedure is to isolate high quality RNA from serum samples. Using our optimized version of the common MRC try reagent RTLS protocol. This is accomplished by first diluting the serum in a one to five ratio with nuclease free water to lower protein contamination.
The second step is to introduce two milliliter phase lock tubes for the first centrifugation step. This traps the majority of contaminants, such as phenol and proteins in the organic phase. Next, the third and final optimization step is the flash centrifugation spin at 12, 000 cheese, which removes any residual contamination from the RNA pellet prior to the ethanol washes.
The final step is to pool two modified RNA preparations from the same patient if a higher total RNA yield is required. Ultimately by using this modified method, which Solubilize serum in nuclease free water employs a phase lock tube to trap major contaminants and introduces a flash spin. We show that it is possible to maximize the recovery of small non-coating RNAs from an RNA isolation from serum.
The main advantage of this technique over existing methods like the standard triol RNA isolation protocol, is that through simple modifications, we can maximize the yield of RNA from serum. Generally, individuals new to this method will struggle because small RNAs are found in low quantities in serum. The optimized method and visualization of these steps will improve recovery of these RNAs and guide novices to a successful isolation Tip.
To begin thaw the previously prepared frozen serum sample on ice, then transfer 400 microliters of the freshly thawed serum into a labeled micro centrifuge tube. Next, dilute the serum with 100 microliters of R nase free water and add 50 microliters of proteinase K at a concentration of one milligram per milliliter. Then incubate the diluted serum at 37 degrees Celsius for 20 minutes to allow adequate protein digestion to ensure complete solubilization at 1.5 milliliters of tri reagent RTLS and 100 microliters of four promo anole.
Briefly invert the homogenate and perform repetitive pipetting for five seconds. Then transfer the mixture into a labeled two milliliter heavy phase lock tube. Spin the homogenate at 12, 000 GS for 20 minutes at four degrees Celsius.
Next carefully decant one milliliter of the resulting aqueous solution into a fresh DNA low bind tube. The organic and interface should be trapped underneath the white gel of the phase lock tube. Then add five microliters of glycogen and 500 microliters of 100%isopropanol to the aqueous solution.
Mix the solution by inversion and incubate it overnight at minus 20 degrees Celsius. Following overnight incubation, centrifuge the sample for 20 minutes at 12, 000 GS in a four degree Celsius centrifuge. Next, discard the clear supernatant and perform a flash spin for two minutes at 16, 000 GS in a four degree Celsius centrifuge.
Using a pipette, carefully remove the clear solution surrounding the pellet. Then wash the pellet with one milliliter of 70%ethanol and centrifuge the sample at 10, 000 GS for 10 minutes. To visualize the pellet, tilt the tube in front of a dark background and decamp the wash solution.
Repeat the wash. Step up. We suspend the pellet in 10 microliters of RNA free water.
Ensure the pellet is completely dissolved by heating the sample at 55 degrees Celsius for five minutes. During this time, mix the sample with repeated pipetting for a higher total RNA yield pool, two RNA preparations from the same patient. Quantitate the resuspended RNA using a UV vs.
Spectrophotometer and assess the RNA quality using a 2100 bioanalyzer. Then store the pooled RNA samples at minus 80 degrees Celsius for use in downstream applications. Spectra photometric profiles of RNA from human serum demonstrate the improved yield of RNA due to the addition of several steps to the traditional chum Chomsky approach.
A typical UV profile of RNA isolated from human serum contrasts with those utilizing optimization steps, including the addition of glycogen and extra spin, and each of those tools combined resulting in a reduction of contaminants and an increased total RNA yield. The purity of the RNA sample was further increased with the use of a phase lock gel, which traps phenol and proteins. Additionally, increasing the volume of serum has a notable impact on RNA yield.
A bioanalyzer trace was performed to further analyze the sample's quality. A single spike at approximately 21 nucleotides represents the micro RNA fraction array profiling and QPCR were performed following the procedure. This array profile of three head and neck cancer and one normal S shows expression of microRNAs, analyze using hierarchical clustering and presented as a heat map where upregulation and downregulation of the microRNAs can be seen.
Following QPCR amplification curves were generated for specific microRNAs. This data was then used to plot raw CT values for the normal serum Once mastered, this technique can be performed across any number of patients producing high quality RNA in today's maximum if it is performed properly Following the procedure. Other methods like quantitative PCR or next generation sequencing can be performed in order to answer additional questions like biomarker discovery and validation.
