The overall goal of this protocol is to quantify small non-coding RNAs on purified lipoproteins. This protocol can help answer key questions in the lipoprotein field, such as defining lipoprotein small RNA signatures in health and disease, as well as advancing lipoprotein small RNAs as disease biomarkers, or cell to cell messages. The main advantage of this technique is one can analyze highly pure lipoproteins for small RNA changes in disease or experimental conditions.
The implications of this technique extends for the therapy of cardiometabolic diseases, because extracellular RNAs are a new class of disease biomarkers and lipoprotein small RNA hold exciting potential as drug targets in future studies. Though this method is designed for HDL small RNAs, it can also be adapted for very low density and low density lipoprotein small RNAs. Generally, individuals new to this method may struggle, because of lack of proper equipment and expertise in preparing samples for density-gradient ultracentrifugation.
This protocol is composed of established methods linked together for the quantification of small RNAs on highly pure HDL, using high throughput sequencing, or real time PCR. This video deals with the purification of the HDL. This protocol's gonna be demonstrated by my assistant, Evan Shadari.
To begin, add 90 microliters of 100x antioxidants to nine milliliters of plasma. Then, adjust the plasma's density to 1.025 grams per milliliter, by adding 0.251 grams of potassium bromide to every nine milliliters of plasma. Rock the plasma at room temperature, until all the salt has dissolved.
Then, transfer the plasma to ultra-centrifuge tubes, and make sure to have a layer of bubbles at the top. There should be no air trapped under the plasma. Next, prepare an 18 gauge needle by adding a 90 degree bend one centimeter from the tip, with the beveled side facing up.
Then, using a syringe and the bent needle, carefully place three milliliters of overlay solution number one onto the plasma. Remove the majority of the bubbles on the surface, and leave two to three millimeters between the meniscus and the tube's edge. Then, carefully place the tubes into SW 40 Ti buckets.
Weigh each bucket with the caps, and exactly balance the buckets that will occupy opposite positions in the centrifuge. Next, place the rotor into the ultra-centrifuge, and run it at 274, 400 times G for 24 hours at four degrees Celsius, with the break set intermediately at a value of four. The next day, carefully remove the buckets from the rotor, and lift the tubes from the buckets.
Then, slowly remove two milliliters from the top layer of the overlay, using a syringe and bent needle. This is the VLDL IDL fraction. Then, collect the remaining seven milliliters of plasma, and transfer it to a 15 milliliter conical tube.
Bring the volume of the plasma up to nine milliliters by adding overlay solution number two. Then, adjust the density to 1.080 grams per milliliter by adding 0.746 grams of potassium bromide. Next, gently rock the sample to dissolve the salt, which will take about 20 minutes.
Then, transfer the sample to an ultra-centrifuge tube without trapping any air. Ensure that a layer of bubbles is made at the top. Using the bent needle, carefully overlay three milliliters of overlay solution number three.
Then, remove the majority of the bubbles and carefully load the tubes into SW 40 Ti bucket. Balance them and spin them, as previously described, for 24 hours. The next day, carefully remove two milliliters from the top layer of the overlay using the bent needle.
This is the LDL fraction. Then, transfer the remaining sample to a new 15 milliliter conical tube, and bring the volume up to nine milliliters with solution number four. Now adjust the density to 1.30 grams per milliliter by adding about 3.33 grams of potassium bromide and rocking the sample until the salt dissolves.
Then, transfer the sample to an ultra-centrifuge tube and carefully overly three milliliters of solution number five. Now, ultra-centrifuge the sample as before, but for 48 hours. Two days later, collect the top two milliliters of the overlay using a syringe and bent needle.
This constitutes the HDL fraction. To remove the high salt solutions, transfer the HDL fraction to a wet dialysis sleeve with a 10, 000 molecular weight cutoff. Then, dialyze it for 24 hours in a liter of one XPBS with gentle stirring at four degrees Celsius.
Replace the PBS solution three times over the 24 hour period. Later, determine the protein concentration of the HDL fraction using a BCA method. Extracellular vesicles, or exosomes, have a similar density to small HDL, and thus could be present in the same density fraction as the HDL.
Thus, the HDL fraction requires further purification by FPLC. Immediately prior to using the FPLC, run 1.2 milligrams of the DGUC HDL fraction in 500 microliters of solution through a 0.22 micron centrifugal filter. Collect the filtered sample into an FDLC injection syringe, ensuring that no air bubbles get trapped.
Next, set the FPLC flow rate to 0.3 milliliters per minute with a pressure limit at 2.6 megapascals. Then, equilibrate the columns with 0.2 column volumes of buffer. Now inject the sample with three milliliters of buffer into the FPLC instrument, and start the run.
Collect 1.5 milliliter fractions for a total of 72 fractions. Identify the FPLC fractions corresponding to DGUC HDL by determining total cholesterol levels using a colorimetric kit according to the manufacturer's instructions. Expect to find six to seven FPLC fractions containing DGUC HDL.
Pool all the HDL containing fractions and concentrate them using a 10 kilodalton cutoff centrifugal filter at 4, 000 times G for an hour at four degrees Celsius, or until HDL concentrate is approximately 100 microliters. Then, collect the HDL concentrate and quantify the protein using the BCA method. Later, use the same amount of HDL total protein to isolate RNA.
Human plasma was fractionated by FPLC without the use of density-gradient separation. Six fractions corresponded to HDL and six to very low density lipoprotein. Lipids in black, cholesterol in red, and protein in blue were all measured by colorimetric kits.
By comparison, use of density-gradient ultra-centrifugation before FPLC resulted in finding lipid protein and cholesterol almost exclusively in HDL fractions. From HDL taken from four healthy plasma donors, total RNA was isolated and sequenced using a single read 50 base pair protocol. The distribution of normalized reads illustrates and enrichment of small RNAs around 21 and 34 nucleotides.
Analysis of the library showed 38 percent of the reads were micro RNAs, and 37 percent were derived from TRNAs. Miscellaneous RNAs, ribosomal RNAs, small RNAs, small nucleolar RNAs, and long non-coding RNAs were also present. Once mastered, this technique can be performed within ten days, when done properly.
While attempting this procedure, it is important to remember to validate HDL small RNA changes observed with high throughput sequencing with another method, such as PCR or northern blotting. Following this procedure, other methods, like HDL cholesterol e-flux acceptance capacity and HDL anti-inflammatory SAs, can be performed during the RNA isolation steps and sequencing prep steps to answer additional questions, like determining the correlation between HDL small RNA changes and HDL function. After its development, this technique paved a way for researchers in the fields of lipids and lipoproteins, to study non-cholesterol cargo, particularly nucleic acids and metabolic diseases, for example hypercholesterolemia or diabetes.
And don't forget, working with human plasma can be extremely hazardous, and one should have proper training in bloodborne pathogens, and appropriate personal protective equipment should be worn when performing this technique.
