A protocol for extraction of total RNA from finger stick whole blood samples preserved with Tempus TM solution [version 1; peer review: 2 approved with reservations]

Monitoring of blood transcriptional changes during disease or treatment could improve the understanding of cellular mechanisms associated with that particular condition. This can be achieved through serial sampling of small blood volumes. However, molecular analysis of gene expression from low volume samples remains a challenging task. To address this issue, we have developed a set of standard operating procedures (SOP), starting from collection of small volume blood to measurement of gene expression. Previously we published an SOP for the collection of a small volume of blood via finger stick and stabilization of RNA. The aim of this manuscript is to share a modified Tempus TM solution based RNA extraction method, developed in our lab, for the extraction of total RNA from low volume whole blood samples collected via finger stick.


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 targets 1 .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][5][6][7][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 environment 9 .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][11][12][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 sampling 14 .A study by Robison et al. found that gene expression measured with venous and finger stick blood collection is comparable 15 .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 stabilization 16 .
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 highquality RNA from pediatric samples 17 .Another study shows the feasibility of an RNA extraction protocol from only 70μl of whole blood collected via finger sticks 15 .Krawiec et al. successfully extracted RNA from even smaller blood samples from mouses or rats 18 .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][22][23][24][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™ method 22 .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 earlier 16 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.

Narrative
Tempus TM spin RNA isolation kits were used for extraction of total RNA from whole blood lysate.The standard protocol recommended by the manufacturer is optimized for 3 ml of whole blood preserved with 6 ml of Tempus TM stabilizing solution.Therefore, modifications were made in order to process 50μl of whole blood collected via finger stick and preserved with 100μl of Tempus TM solution.Briefly, whole blood lysate is thawed and washed with 1xPBS (phosphate buffer saline) to obtain the RNA pellet.RNA purification is achived by using RNA purification filters and wash buffers.DNase treatment is performed to remove DNA contamination and finally purified RNA is eluted from the column using elution solution.RNA yields and quality are measured on NanoDrop and Fragment Analyzer.

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 publication 16 .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-seq 26 or RT-PCR 27 , while degraded RNA is suitable as input for the Nanostring assay (samples with RIN as low as 3 28 .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.

Reagents and equipment
• Tempus TM  3. Add 50μl of 1x PBS to each sample and vortex the tubes vigorously for 30 seconds to ensure proper mixing.Then, centrifuge the samples at 4°C at 3,000g for 30 minutes.4. Pour off the supernatant and leave the tubes on absorbent paper for 1-2 minutes.

Add 400μl of RNA Purification Resuspension Solution
to the sample tubes and vortex briefly to resuspend the RNA pellet.Keep the RNA pellet on ice during the preparation for the next steps.
6. Insert the RNA purification filter into waste collection tube and pre-wet it with 100 μl of Wash Solution 1.
7. Add the resuspended RNA to the purification filters and centrifuge for 30 seconds at 16,000g.
8. Remove the purification filters and discard the liquid waste.Re-insert the purification filters into the waste tube.
9. Add 500μl of Wash Solution 1 into the purification filters and centrifuge for 30 seconds at 16,000g.Discard the samples' flow through and re-insert purification filter into the waste tubes.
10. Add 500μl of Wash Solution 2 to the purification filter and centrifuge the sample for 1 minute at 16,000g.
11. Discard the flow through and re-insert the purification filter into the waste tube.Add 100μl of absolute RNA Wash Solution and incubate at room temperature for 15 minutes.
12. Add 500μl of Wash Solution 2 into the purification filter, incubate at room temperature for 5 minutes and centrifuge for 30 seconds at 16,000g.13.Perform an additional wash with 500μl of Wash Solution 2. Discard the flow through and re-insert the purification filter into a clean collection tube.

Sunil M. Kurian
The Scripps Research Institute, San Diego, CA, USA This article presents a modified version of a finger stick method of RNA extraction using an already-established Tempus system for RNA.While the study is relevant especially for quick and easy collection of RNA in specific cases such as pediatric samples as well as samples that have to be collected in large populations in a quick and reliable way, I do have some questions and clarifications regarding the study: While they have been through specific differences between the Tempus and the PaxGene systems for RNA extraction, in this study with the modified methodology there have been no specific attempts to do downstream processing in terms of RNA sequencing or microarray analysis to compare the results.It is important that the authors do not just assume that previous results may replicate despite modifications to the protocol.A quick comparison using a microarray, or a targeted RNAseq method would actually sort out the true differences between the modified method and a standard PaxGene or a PaxGene methodology that has been modified for finger stick purposes. 1.
As a follow-up to the first question, a downstream processing step to compare RNA quality becomes even more important in the context of having a range of RNA Integrity numbers.Do higher quality numbers translate to better QC when it comes to sequencing or microarrays?These answers will shed more light into the actual quality of RNA from this modified method.

2.
Have the authors tried to do a globin reduction step to remove globin transcripts which are highly abundant and may interfere with the quality of the other expressed RNA transcripts?What is their opinion about performing a globin reduction step?Many protocols suggest this step when dealing with whole blood samples, so it would be nice to get some input about it.

3.
Some comments on the wide range of RNA yields obtained need to be explained.Do the authors expect such a wide range in total RNA to be normal?Is it more of a technical limitation of the methodology that scales down a protocol that normally uses a few mls of whole blood to microliters of blood?What is the experience from their previous studies?

4.
Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Organ Transplant, Genomics, Biomarkers I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.
The benefits of publishing with F1000Research: Your article is published within days, with no editorial bias • You can publish traditional articles, null/negative results, case reports, data notes and more • The peer review process is transparent and collaborative • Your article is indexed in PubMed after passing peer review • Dedicated customer support at every stage • For pre-submission enquiries, contact research@f1000.com

Figure 1 .
Figure 1.Analysis of quality of total RNA on Fragment Analyzer.Representative electrophorograms used for RNA quality analysis on Fragment analyzer.

Table 1 . Quality control analysis of total RNA extracted from finger stick whole blood samples.
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.
This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.