Isolation of Small Noncoding RNAs from Human Serum

Summary

This protocol describes a method for extracting small RNAs from human serum. We have used this method to isolate microRNAs from cancer serum for use in DNA arrays and also singleplex quantitative PCR. The protocol utilizes phenol and guanidinium thiocyanate reagents with modifications to yield high quality RNA.

Abstract

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are found in mammalian cells. These small RNAs are potent gene regulators controlling vital pathways such as growth, development and death and much interest has been directed at their expression in bodily fluids. This is due to their dysregulation in human diseases such as cancer and their potential application as serum biomarkers. However, the analysis of miRNA expression in serum may be problematic. In most cases the amount of serum is limiting and serum contains low amounts of total RNA, of which small RNAs only constitute 0.4-0.5%¹. Thus the isolation of sufficient amounts of quality RNA from serum is a major challenge to researchers today. In this technical paper, we demonstrate a method which uses only 400 µl of human serum to obtain sufficient RNA for either DNA arrays or qPCR analysis. The advantages of this method are its simplicity and ability to yield high quality RNA. It requires no specialized columns for purification of small RNAs and utilizes general reagents and hardware found in common laboratories. Our method utilizes a Phase Lock Gel to eliminate phenol contamination while at the same time yielding high quality RNA. We also introduce an additional step to further remove all contaminants during the isolation step. This protocol is very effective in isolating yields of total RNA of up to 100 ng/µl from serum but can also be adapted for other biological tissues.

Introduction

In recent years, there has been a growing push to discover novel biomarkers for the early detection of human diseases. Much attention has been focused on using small RNAs such as microRNAs² (miRNAs or miRs) as potential markers. These small RNAs are found in body fluids such as serum and studies have shown they are resilient to degradation and are stable over a range of varying environmental conditions³. Given these features, serum or circulating miRNAs are the ideal biomarker^4,5. Currently there are two main approaches for the isolation of small RNA from biological fluids. The first approach uses column-based technology to bind and elute the small RNAs⁶, while the second approach uses the long-standing protocol with phenol and guanidinium thiocyanate reagents⁷. We have developed a simple, effective, column-free protocol to isolate small RNAs from human serum. The isolated RNA is immediately usable in downstream applications, including DNA oligonucleotide arrays and RNA sequencing.

This protocol was developed because we were confronted with several issues when using phenol-based methods to isolate RNA from serum. The traditional Chomczynski approach is frequently used in most laboratories with a range of reagents available from most commercial vendors. However considering their widespread use, stringent guidelines have not been developed to consistently produce high quality RNA from bodily fluids, in particular blood or serum.

Common problems associated with isolating RNA from serum include low RNA yields and contamination with reagents used during the isolation, particularly phenol. Our approach eliminates these phenol contaminants to provide high quality RNA for downstream analysis such as quantitative PCR (qPCR) and RNA sequencing. We have further tested this RNA on miRNA arrays.

Protocol

Note: Human serum samples from healthy patients or patients with cancer were obtained with informed consent under approved human ethical protocols from the Royal Prince Alfred Hospital Sydney (Protocol number X10-0016 and HREC/10/RPAH/24) and the University of Technology, Sydney. Serum samples were collected from patients before surgery from various Sydney hospitals and placed in storage at -80 °C.

1. Small RNA Isolation from Serum 

Total RNA was prepared from human serum using a modified version of the Tri-Reagent RT LS protocol.

1. Thaw frozen serum sample on ice and then transfer 400 µl of the freshly thawed serum into a labeled microcentrifuge tube.
2. Dilute the serum with 100 µl of RNase free H₂O and add proteinase K at a concentration of 1 mg/ml.
3. Incubate at 37 °C for 20 min to allow protein digestion by proteinase K.
4. To ensure complete solubilization, add 1.5 times its volume of Tri-Reagent RT LS and 100 µl of 4-bromoanisole.
5. Briefly invert the homogenate, perform repetitive pipetting for 5 sec and transfer into a labeled 2 ml Heavy Phase Lock tube.
6. Spin the homogenate at 12,000 × g for 20 min at 4 °C.
7. Carefully decant at least 1 ml of the resulting aqueous solution into a fresh DNA Lobind tube. The organic and interphase should be trapped underneath the white gel of the Phase Lock tube.
8. Add 5.0 µl of glycogen (5 mg/ml) and 500 µl of 100% isopropanol to the aqueous solution, mix by inversion, and incubate O/N at -20 °C.
9. Following O/N incubation, centrifuge the sample for 20 min at 12,000 × g in a 4 °C centrifuge.
10. Discard the clear supernatant and perform a "flash" spin for 2 min at 16,000 × g in a 4 °C centrifuge.
11. Carefully remove the clear solution surrounding the pellet using a pipette.
12. Wash the pellet with 1 ml of 70% ethanol and centrifuge at 10,000 × g for 10 min. Decant the wash solution and repeat wash step.

2. RNA Resuspension into Solution

1. Resuspend the pellet in 10 µl of RNase free H₂O, ensuring the pellet is completely dissolved. To ensure that the RNA is completely solubilized the sample can be heated to 55 °C for 5 min. During this time, mix the sample with repeated pipetting. For a higher total RNA yield, pool two RNA preparations from the same patient.
2. Quantitate the resuspended RNA using a UV-Vis spectrophotometer and assess the RNA quality using a 2100 Bioanalyzer. Store the pooled RNA samples at -80 °C for use in downstream applications.

Results

Figure 1 represents a typical UV/Vis spectrum of RNA isolated from serum. From this profile we noted protein contamination at 280 nm with phenol and organic contaminants both at 270 nm and 230 nm, respectively. Residue guanidinium thiocyanate was also noted at 260 nm. To reduce contaminants, a series of optimization steps were made to the standard Tri-Reagent RT-LS procedure. We added 5 mg/ml of glycogen to increase both the total RNA yield and reduce contamination (black line, Figure 1A...

Discussion

The population of microRNAs constitutes approximately 0.4-0.5% of the total RNA found in serum. Further there is also a high protein content found in human serum. To improve RNA and reduce both protein and phenol contaminates we have modified the traditional Chomczynski approach⁹ with the addition of several steps.

Total RNA was isolated from serum using the standard Tri-Reagent RT-LS (Molecular Research Centre) however when performed in our laboratory, this method yielded RNA in lo...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Tri-Reagent RT LS	Molecular Research Center, USA	TR 118
RNase free H2O	GIBCO Invitrogen	10977-023
Proteinase K	Finnzymes, Finland	EO0491
Heavy Phase Lock tube	5PRIME	2302830	2 ml capacity
DNA Lobind tube	Eppendorf	0030 108.078	1.5 ml capacity
Glycogen	Invitrogen, USA	10814-010	5 mg/ml
RNA grade isopropanol	Sigma Aldrich, USA	I9516	100%
Refrigerated centrifuge	John Morris
Nanodrop UV-Vis spectrophotometer	Thermo Fisher Scientific, USA
RNA grade ethanol	Sigma Aldrich, USA	E7023	70%
Agilent 2100 Bioanalyzer	Agilent, USA