A protocol for extraction of total RNA from finger stick whole blood samples preserved with Tempus^TM solution

Introduction

The transcriptome is the complete set of transcripts in a specific type of cell or tissue. Transcriptome datasets can be leveraged to understand the genes or pathways associated with particular conditions which will help to develop diagnostic biomarkers and to identify new therapeutic targets¹. Although transcriptional profiles of target diseased tissues or cells are ideal biotypes for such analyses, procuring tissue biopsies/cells and extracting a sufficient amount of RNA from these specimens prove often impossible in clinical settings. Therefore, whole blood is often considered as an alternative surrogate tissue in clinical research^2,3. Blood plays a crucial role in immunity, inflammation and physiological homeostasis. Blood-based profiles also constitutute a powerful means for exploring basic biology and for approaching the complexity of biological systems.

The rapid advances in transcriptome profiling technologies, such as microarray and next-generation sequencing, made it possible to measure simultaneously the abundance of RNA on a genome-wide scale. High throughput RT-PCR and NanoString offer the opportunity to profile hundreds of targets at a lower cost than sequencing technologies. Overall, practicality as well as affordability allow studying changes in blood transcript abundance in infection, treatment or specific conditions and enable to maximize information obtained from each patient. Correlating serial blood transcriptome markers with the clinical course of disease has been shown to be a potential approach in diagnostics and for assessment of treatment response^4–8.

Minimization of the technical variance in any assay plays a critical role in the measurement of true biological variance. In transcriptome studies, the sources of technical variance can be considerable. In particular, RNA isolation and purification steps greatly influence the results of gene expression profiling, since RNA is a highly unstable molecule that is easily degraded by RNases which are ubiquitous in the environment⁹. Therefore, extra care has to be taken during this process. Furthermore, the protocol used for the extraction of RNA should (i) provide quantitative recovery of RNA that is intact and free from contaminants and (ii) keep the sample as concentrated as possible for further downstream analysis. There are commercial RNA whole blood collection tubes available in the market; PAXgene™ Whole Blood RNA isolation system (Qiagen, Germany) and Tempus™ Whole Blood RNA isolation system (ThermoFisher Scientific, USA). They have a significant advantage as they lyse whole blood at the time of collection, while simultaneously stabilizing RNA for later purification. However, these collection systems require drawing of 2.5 ml to 3 ml of venous blood at each collection time point, which can be challenging in some settings (e.g. pediatric populations, high frequency collection, home self-collection, field collection).

Finger-stick blood collection is a widely used and safe method for the collection of blood, especially when only small volumes are required^10–13. Major advantages of this collection method are that it is less invasive, quicker and can be performed without a trained phlebotomist. Therefore, it is more amenable to field applications and repeated sampling¹⁴. A study by Robison et al. found that gene expression measured with venous and finger stick blood collection is comparable¹⁵. However, currently available microtainers used for collection of whole blood samples via finger stick methods do not contain any RNA stabilizing solutions. Therefore, this method requires modified protocols for collection of blood, stabilization of samples and extraction of RNA. Recently, we published a detailed a standard operating procedure (SOP) for finger stick blood collection and RNA stabilization¹⁶.

There are few published reports which describe the procedure for extraction of RNA from a small volume of whole blood. A study by Carrol et al. used small volumes of blood (≥300μl) along with a modified PAXgene protocol to obtain high-quality RNA from pediatric samples¹⁷. Another study shows the feasibility of an RNA extraction protocol from only 70μl of whole blood collected via finger sticks¹⁵. Krawiec et al. successfully extracted RNA from even smaller blood samples from mouses or rats¹⁸. However, all these studies used PAXgene based protocols for the purpose of extracting total RNA. Reported yields and quality of RNA stabilized in Tempus^TM solution was generally greater when compared to PAXgene solution^6,19,21–25. When PAXgene™ and Tempus™ were compared by using microarrays as the readout, several known phytohemagglutinin (PHA) inducible genes were only found to be up-regulated when RNA was isolated using the Tempus™ method, but not using the PAXgene™ method²². Considering these factors, we have developed a modified Tempus^TM solution-based RNA extraction method for the extraction of total RNA from low volume whole blood samples (50μl of whole blood). This method is currently employed in the context of a pregnancy monitory study being conducted on the Thai-Myanmar border (manuscript in preparation). The study aims to assess transcriptional changes in women during pregnancy and in the mother and child post-partum. Overall ~20,000 whole blood samples will be collected from 400 mother and child pairs. The low volume blood sample collection and RNA stabilization method published earlier¹⁶ and the related RNA extraction method described below are being shared with an anticipation that they may be of use to others and be improved through comments from reviewers and readers.

Narrative of the procedure

The procedure described in this article can be used for extraction and quality control of total RNA from a small volume of whole blood preserved with Tempus^TM solution. A narrative is provided in this section, along with general remarks and considerations. A detailed point-by-point SOP follows.

General remarks

1) Blood sample collection, storage and shipment

The detailed protocol for collection and storage of whole blood samples for gene expression studies is available in our recent publication¹⁶. Briefly, 50μl of blood samples are collected using a plastic capillary straw in a microcentrifuge tube. Following thorough mixing with 100μl of Tempus^TM RNA stabilizing solution it is stored at -20°C, preferably, or alternatively -80°C. The transcriptional profile is maintained due to effective stabilization of RNA and will accurately reflect the physiological state of the patient at the time of the blood draw. As mentioned above, this sampling protocol is being used for one of our studies which investigates alteration in temporal transcriptional and microbiome trajectories preceding pre-term birth. For this study, blood samples are collected in Thailand and transfered to Qatar on dry ice. RNA yields and integrity reported below indicate that this shipment method permits recovery of nucleic acid in quantities and quality that meet requirements of downstream applications such as RNAseq or PCR.

2) RNA extraction protocol

This protocol is developed by incorporating the following modifications to the standard Tempus™ Spin RNA isolation protocol for the extraction of total RNA from Tempus^TM cell lysate:

(a) Washing with PBS

Add 5μl of 1x PBS to the 150μl of Tempus^TM lysate, vortex, and centrifuge. After washing, resuspend the RNA pellet in 400μl of RNA Purification Resuspension Solution.

(b) DNase treatment

Perform the DNase treatment as per the recommended protocol. This is an optional step in the manufacturer’s recommended protocol.

(c) Incubation with Nucleic acid purification elution solution

Add Nucleic Acid Purification Elution Solution to the samples and incubate at 70°C for 1 minute. In our lab, extending the incubation time, for instance to 2–5 minutes, did not help to improve RNA yield.

(d) Elution of RNA

At the final step, elute the purified RNA with either 25μl or 50μl of elution solution. The concentration of eluted RNA will range from 5 to 20ng/μl with 50μl of elution solution and will be >10ng/μl with 25μl of Elution Buffer.

3) RNA yields and quality

In a set of 25 whole blood samples >200ng of total RNA could be extracted from 50μl of whole blood (mean±standard deviation: 503±170 ng; Range: 228–861 ng). The RNA integrity numbers (RIN) ranged from 5.9 to 9.2. (mean±standard deviation: 7.5±0.7). These figures are compatible for downstream applications such as RNAseq (input RNA: 150–200 ng), RT-PCR (input RNA for cDNA synthesis - depending on the kit: 10–500 ng) and NanoString (input RNA as little as 10 ng). Samples with an RIN > 5.3 is shown to be sufficient for downstream applications such as RNA-seq²⁶ or RT-PCR²⁷, while degraded RNA is suitable as input for the Nanostring assay (samples with RIN as low as 3²⁸. Table 1 and Figure 1 show the quality control analysis of total RNA extracted from selected whole blood samples. It is important to note that this method may not be used for extraction of small RNA such as miRNA since smaller RNAs (i.e. <200nt) are washed off during purification step. Alternative options for RNA extraction methods allowing retention of miRNAs include Norgen (Norgen Biotek Corporation) and MagMAX (ThermoFisher Scientific) RNA extraction kits, as they claim to provide suitable solutions for extraction of all sizes of RNA, from the large mRNA and ribosomal RNA down to microRNA.

Table 1. Quality control analysis of total RNA extracted from finger stick whole blood samples.

S. No	Sample ID	Concentration of total RNA (ng/ul)	Volume of elution buffer (ul)	Quantity of total RNA (ng)	A260/280	RIN
1	MSP-002-Blood-001	22.58	25	565	1.71	6.9
2	MSP-002-Blood-007	16.05	25	401	2.08	8.3
3	MSP-002-Blood-013	16.28	25	407	1.87	8.0
3	MSP-007-Blood-006	14.81	25	370	2.15	9.2
4	MSP-007-Blood-012	17.23	25	430	2.08	5.9
5	MSP-011-Blood-001	24.52	25	613	2.07	6.8
6	MSP-011-Blood-006	14.76	25	369	2.05	6.7
7	MSP-011-Blood-012	21.55	25	538	1.75	6.8
9	MSP-019-Blood-007	13.23	25	330	1.99	7.7
10	MSP-019-Blood-013	30.75	25	768	2.25	8.5
11	MSP-002-Blood-003	5.36	50	268	2.12	6.0
12	MSP-002-Blood-005	5.77	50	289	1.91	7.3
13	MSP-002-Blood-008	10.72	50	536	1.82	7.3
14	MSP-002-Blood-014	15.29	50	765	1.75	7.7
15	MSP-002-Blood-015	12.87	50	643	1.8	7.0
16	MSP-007-Blood-004	4.57	50	228	1.82	7.1
17	MSP-007-Blood-009	6.72	50	336	1.83	7.6
18	MSP-007-Blood-013	17.22	50	861	1.82	7.4
19	MSP-007-Blood-014	14.87	50	744	1.75	7.5
20	MSP-011-Blood-003	12.52	50	626	1.82	8.1
21	MSP-011-Blood-002	12.48	50	624	1.73	7.6
22	MSP-011-Blood-009	9.63	50	481	1.85	8.0
23	MSP-011-Blood-010	9.07	50	453	1.86	7.6
24	MSP-011-Blood-011	9.22	50	461	1.75	7.7
25	MSP-011-Blood-013	9.20	50	460	1.89	8.2

Blood samples (50ul) were collected from pregnant women via a finger stick. Total RNA was extracted using Tempus spin RNA extraction kit. At the end, purified RNA was eluted with 25ul or 50ul of elution buffer. A260:280 ratio was measured in Nanodrop. RNA integrity Number (RIN) was measured on Fragment Analyzer.

Figure 1. Analysis of quality of total RNA on Fragment Analyzer.

Representative electrophorograms used for RNA quality analysis on Fragment analyzer.

Materials and methods

Data availability

All data underlying the results are available as part of the article and no additional source data are required.