How Long Does the mRNA Remains Stable in Untreated Whole Bovine Blood?

doi:10.21203/rs.3.rs-434702/v1

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How Long Does the mRNA Remains Stable in Untreated Whole Bovine Blood?

https://doi.org/10.21203/rs.3.rs-434702/v1

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High quality and quantity of messenger RNA (mRNA) are required for accuracy of gene expression studies and other RNA-based downstream applications. Since RNA is considered a labile macromolecular prone to degradation, which may result in falsely altered gene expression patterns, several commercial stabilizing reagents have been developed aiming to keep RNA stable for long period. However, for studies involving large number of experimental samples, the high costs related to these specific reagents may constitute a barrier. In this context the present study was designed aiming to evaluate the stability of mRNA in whole bovine blood collected in K3EDTA tubes during storage at common fridge (4°C). Whole blood samples were collected from four Holstein cattle, and RNA extractions were performed immediately after blood sampling (< 2 h) and at 1-day post-sampling (dps) and at 2 dps, 3 dps, 7 dps and 14dps intervals. RT-qPCR assays were run using eight different genes (B2M, ACTB, PPIA, GAPDH, YWHAZ, CD4, TNF-α and IFN-γ) aiming to evaluate the presence of altered gene transcription during storage. All extracted RNA samples presented high purity and integrity, and there were no significant differences of gene expression values between 2hps to 2 dps intervals for all evaluated genes. Bovine blood RNA remained stable in K3EDTA tubes for 2 days stored at common fridge and can be successfully and accurately used for gene expression studies.

Molecular Biology

RNA integrity

gene expression

cattle

stability

storage

fridge

Messenger RNA levels may constitute good indicator of physiological and/or immune status of cells, addressing the host responses under different environmental conditions, targeting the better understanding of mechanisms of host-pathogen interactions.

Several studies targeting the quantification of gene expression profiles have been performed to better understand human and veterinary diseases (Hammerle-Fickinger et al., 2009; Sheridan et al., 2012; Brym et al., 2013; Caetano et al., 2019; Donohue et al., 2019; Gautam et al., 2019; Garner et al., 2020; Rodríguez et al., 2020). In this context, blood specimens are the most common applied matrix, since peripheral blood may be considered less invasive and valuable source of RNA in vertebrates (Liu et al., 2015). However, RNA blood is highly vulnerable to degradation, which may consequently influence on the results of RNA studies to varying levels (Liu et al., 2015).

The appropriated storage of samples, before RNA extraction procedure, is of utmost importance in order to keep the RNA quality. Therefore, different methods have been applied to avoid RNA degradation. Snap frozen using liquid nitrogen (-180°C) is one of the most common ways, though cryopreservation is not always possible or practical. Stabilizing reagents are also commonly applied, such as PAXgene blood RNA tubes (PreAnalytix, Qiagen/BD, Hombrechtikon, Switzerland), RNAprotect Animal Blood Tubes (Qiagen, Venlo, Netherlands), Tempus Blood RNA tubes (Applied Biosystems, Foster City, CA) and RNAlater stabilization reagent (Thermofisher, Waltham, MA). Although, all these reagents, except for RNAlater reagent, require proper commercial RNA extraction kits, and the costs related to these reagents may be very expensive, especially for experiments including a large sampling size.

A second important drawback associated to most of these blood collection devices is due to the high amounts of globin mRNA, released from reticulocytes during total cell lysis immediately after sample collection, which may lead to decreased RT-qPCR detection sensitivity of rare mRNA transcripts and/or increases of signal variation on microarrays (Wright et al., 2008). Additional globin depletion steps were performed to circumvent these problems, but this strategy was pointed out as inefficient, besides the requirement of related new costs (Liu et al., 2006). Finally, most of these reagents are also not applicable for studies targeting specific cell subsets, since all blood cells are lysed when added to the stabilizing reagent.

Different pre-treatment methods for RNA extraction were compared for human blood specimens, from whole blood or buffy coat isolation, followed or not by RNA stabilization with Trizol Reagent, TRizol LS Reagent, RNAlater or Paxgene Blood RNA tubes, wherein the method using a buffy coat pre-separation added directly to Trizol Reagent yielded the highest RNA integrity and quality for RNA extraction, emphasizing here the superior quality compared to pre-treatments with RNAlater or Paxgene Blood RNA tubes (Liu et al., 2015).

Regarding the stabilization of cellular RNA in blood stored at room temperature, some studies have demonstrated RNA degradation in whole blood samples longer than 32h, but when whole blood samples were stored at low temperature (4°C), the RNA integrity remained stable at duration of 40 hours (Huang et al., 2017). Since transcription and translation can continue after collection of samples, the final RNA composition may present differences compared to RNA content at the moment of collection (Rodriguez et al., 2020). In this context, altered transcription levels were also observed in whole blood samples stored at room temperature after 24 hours (Das et al., 2014, Huang et al., 2017) or at low temperature (Huang et al., 2017).

Most of studies targeting RNA stabilization in whole blood samples were performed in human specimens, and to our knowledge there are no similar investigations for bovine whole blood. Thus, the aim of this study was to evaluate the storage time that RNA remains stable in bovine whole blood, regarding purity, integrity and unaltered gene transcription profiles at common fridge (4°C).

Sample collection and storage intervals in common fridge

Twenty-four blood samples (six samples per animal) were collected from the jugular vein from four Holstein calves, using a vacuum system (Vacutainer®, Becton Dickinson) containing EDTA anticoagulant. After collection, the blood samples were stored in a refrigerator at 4 ° C until the moment of analysis. This study was approved by the Ethics Committee on Animal Experimentation of the Instituto de Zootecnia (Protocol Nr. 298-2020).

Extraction of total RNA from whole blood

The Twenty-four blood samples were divided into six groups and were submitted to RNA extraction the following five-time intervals post-collect: 2 hours, 1 day, 2 days, 3 days, 7 days and 14 days. The RNA extractions were performed using the protocol described by Jiang et al., (2013), with some modifications. Each tube containing 5 mL of whole blood was centrifuged at 2000 x g at 4 °C for 10 min. The layer of plasma was discarded, and 0.5 mL of buffy coat layer was aspirated, transferred into a new 2 mL microtube and 1.5 mL of RBC lysis buffer was added. The tube was vigorously shaken and incubated at room temperature (RT) for 15 min prior to centrifugation at 2,000 x g for 10 min (4°C). Supernatant was discarded, and 1.0 mL RBC lysis buffer was added to the cell pellet, followed by incubation at RT for 10 min. After centrifugation at 2,000 x g for 10 min, at 4 °C, supernatant was discarded, WBC pellet lysed and homogenized with 1.0 mL Qiazol® Reagent (Qiagen, Hilden, Germany). The lysate was transferred into a 1.5 mL microtube and incubated at RT for 5 min. Then 0.2 mL of 1-Bromo-3-chloropropane was added to the lysate, followed by vigorously shaken for 15 sec and incubated at RT for 5 min. The sample was centrifuged for 15 min at 12,000 x g at 4 °C. The upper layer was transferred to 1.5 mL fresh tube, mixed with 0.5 mL 100% isopropanol alcohol, and incubated at RT for 10 min. The mixture was centrifuged for 10 min at 12,000 x g at 4 °C. The pellet was washed with 1 mL 75% ethanol and centrifuged at 7,500 x g for 5 min. at 4 °C. The pellet was air dried and dissolved in 50 μL TE-buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, pH 8.0). The recovered RNA was stored at –80°C.

The concentration and purity of the RNA samples were determined using a BioDrop spectrophotometer (BioDrop uLITE, Integrated Scientific Solutions Inc., Walnut Creek, CA, USA) and subjected to electrophoresis on a 2% agarose gel stained with ethidium bromide and visualized on the BioRad Gel DocTM XR+ imager (Bio-Rad Laboratories, Hercules, CA, USA).

DNase digestion and cDNA synthesis

The cDNA synthesis was performed using the High-Capacity cDNA Reverse Transcription kit with RNase inhibitor (ref. No. 4374967, Applied Biosystems, CA, USA), following the manufacturer's information, using OligodT primers (IDT, Coralville, IA, USA). Previously, RNA samples were treated with RQ1 RNase-Free DNase (Promega, Wisconsin, US, cat. # M6101), following the manufacturer's recommendations. In addition to the RNase treatments, the absence of genomic DNA amplification in the RNA samples was confirmed by qPCR assays containing treated RNA instead cDNA.

Primer design and analysis of the genes

The primers for all evaluated genes were designed using the PrimerQuest software (http://www.idtdna.com/Primerquest/Home/Index). The specificities and qualities of the primers were tested using an online tool NetPrimer (http://www.premierbiosoft.com/netprimer/), OligoAnalyzer IDT (https://www.idtdna.com/calc/analyzer) and BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGETYPE=BlastSearch&LINKLOC=blasthome) and Primer-Blast (https://www.ncbi.nlm.nih.gov/tools/primer-blast/). Details of specific primer sets are shown in Supplementary information 1.

The expression profiles of eight genes were evaluated: Beta-actin (ACTB) which encodes one of the two non-muscle cytoskeletal actins, Beta-2-microglobulin (B2M) which encodes a component of MHC I molecule, Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) which encodes an enzyme of glycolysis pathway, Peptidylprolyl isomerase A (PPIA) encodes a protein responsible by catalyzes of the cis-trans isomerization of proline imidic peptide bonds involved in many biological processes (intracellular signaling, transcription, inflammation and apoptosis), Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta (YWHAZ) which encodes a central hub protein for many signal transduction pathways, cluster of differentiation 4 (CD4) which encodes a molecule expressed in the surface of T cells, macrophages, monocytes, dendritic cells and neutrophils, Interferon-gama cytokine (IFN-γ) which encodes a soluble cytokine member of type II class of interferons and Tumor Necrosis Factor alpha (TNF-α) which encodes a pro-inflammatory cytokine. The algorithms used to identify the most stable genes were geNorm (Vandesompele et al., 2002), NormFinder (Andersen et al., 2004) and BestKeeper (Pfaffl et al., 2004). In addition, a RankAggregated weighted classification aggregation tool (Pihur et al., 2007) was used to determine a general ranking of the most stable genes.

RT-qPCR assay

The qPCR reactions were carryout on the Rotor gene Q equipment (Qiagen, Hilden, Germany) for a final volume of 10 mL that contained: 5 mL of the QuantiNova SYBR® Green RT-PCR Kit (Qiagen) master mix, 0.3 mL (10 mM) of each primer, 2.4 mL of ultra-pure water (Sigma-Aldrich) and 2 mL of cDNA. One cycle of enzymatic activation of 95 ° C was programmed for 2 minutes followed by 35 cycles of 95 ° C for 10 seconds and 60 ° C for 30 seconds. After amplification, a melting temperature analysis was generated by raising the incubation temperature from 60 °C to 95 °C in 0.5 °C increments with a hold step of 5 sec at each increment. All reactions were run in duplicate.

Statistical analysis

Statistical analysis was performed using a mixed model described by Steibel et al (2009) to jointly analyze the expression of target and reference genes [y_gijkr = TG_gi+ G_gj + D_ijk + e_gijkr].

The model was adjusted using the "Mixed" procedure of the SAS (SAS Institute, Cary, NC). The comparisons between treatments were made through the assembly of contrasts according to Steibel et al. (2009) that allowed the hypotheses to be tested at values equivalent to -ΔΔCq. Comparisons were performed between 2hps and other intervals, aiming to evaluated if storage time led to altered gene expression.

RNA quality and integrity during blood storage at common fridge

The total RNA concentration and A_260/280 ratio averages were 939.23 ± 246.25 ng/µL 1.92 ± 0.04. The results of RNA concentration and A_260/280 ratios are present in Table 1. Regarding RNA concentrations and A_260/280 ratio means, the difference was only found between the first extraction (2 h) and the 3 days. Same difference was also observed for results related to the 260/280 and 260/230 nn ratios (Table 1). The integrity of all RNA samples extracted between all intervals were also observed in the gel electrophoresis (Fig. 1). The 18S and 28S ribosomal RNA bands were clearly visible with no smeared bands, indicating presence of high integrity of RNA sample.

Table 1

Means (± SD) of RNA and cDNA concentrations (ng/µL) and RNA and cDNA purity levels estimated by 260/280 nm ratio readings.
	RNA			cDNA
Interval	[ ] ng/mL	Ratio 280/260	Ratio 260/230	[ ] ng/mL	Ratio 280/260	Ratio 260/230
2 h	924.00 ± 107.23bc	1.94 ± 0.01ab	2.06 ± 0.12a	2392.75 ± 39.36a	1.72 ± 0.01a	2.19 ± 0.02ab
1 dps	1028.80 ± 124.93ab	1.96 ± 0.01a	1.96 ± 0.09a	2389.25 ± 49.04a	1.71 ± 0.01a	2.18 ± 0.01ab
2 dps	918.00 ± 136.28bc	1.95 ± 0.01a	1.83 ± 0.10ab	2353.75 ± 23.19a	1.72 ± 0.01a	2.19 ± 0.01ab
3dps	1296.20 ± 184.33a	1.87 ± 0.01c	1.41 ± 0.19b	2423.25 ± 49.39a	1.71 ± 0.01a	2.14 ± 0.03b
7 dps	859.00 ± 242.93bc	1.92 ± 0.03ab	1.84 ± 0.26ab	2416.50 ± 72.73a	1.71 ± 0.01a	2.16 ± 0.04ab
14 dps	609.40 ± 106.25c	1.90 ± 0.03bc	1.78 ± 0.33ab	2320.50 ± 59.75a	1.75 ± 0.01a	2.20 ± 0.03a
Means followed by different lowercase letters in the same column differ significantly (p ≤ 0.05)

Gene regulation

The B2M and PPIA genes were the most stable, while GAPDH and ACTB were considered the least stable genes (Supplementary information 2).

There was no altered expression for all evaluated genes between samples stored for 1 and 2 days compared to the samples processed immediately after sampling (2hps). Altered gene expression were observed for: ACTB at 3dps and 14 dps, CD4 at 7dps and 14 dps, YWHAZ and TNF-a at 14 dps (Figure 2).

The accurate transcriptional quantification and high reproducibility of RT-qPCR assays are crucial for the understanding of biological regulation when gene expression studies are performed. Moreover, proper standardization of blood sample handling and posterior procedures are also required for those experiments. Therefore, the present study aimed to evaluate the storage time that RNA remains stable in bovine whole blood samples without stabilizers addition, regarding purity, integrity and unaltered gene transcription profiles at common fridge (4°C).

The extraction method was able to provide high yielded RNA, presenting high purity and integrity, from whole blood samples stored during all the experimental period (from 2 hours to 14 days). Regarding the RNA concentrations, there were significant differences between intervals, although the values between < 2 h and 14 days did not differ significantly. Since the WBC was obtained by manual pipetting after centrifugation, these differences can be expected, since different amounts of WBC could be aspirated. The RNA extraction method used in the present study was based on the methodology described by Jiang et al., (2013), with some modifications. These authors compared three methods of RNA extraction from blood samples and verified that the extraction method also applied in the present study produced the highest yield, whereas lower yields were obtained by the other two methods. Another advantage of specific processing of WBC is associated to decreased globin interference effects on PCR sensitivity. In this context, several studies have demonstrated that the abundance of globin genes in whole blood may mask the underlying biological differences between whole blood samples (Liu et al., 2006; Field et al., 2007; Li et al., 2008; Wright et al., 2008; Raghavachari et al., 2009).

The RNA integrity was maintained during the 14 days of whole blood samples storage at common fridge. Narrow variation of Cq values were observed for all evaluated genes during RT-qPCR assays, therefore, we assumed that RNA samples isolated from whole bovine blood stored at common fridge up to 14 days can be used for RNA sequencing purposes. On the other hand, since altered gene expression results were observed from 3dps, we concluded that whole blood samples can be stored up to 2 dps for studies targeting gene expression profiles.

There are scarce studies available focused on assessing the RNA integrity and gene expression during storage of whole blood samples. To our knowledge this was the first report to evaluate the storage time that RNA kept stable in untreated whole bovine blood at common fridge, without addition of any RNA stabilizing reagents, and may constitute a valuable information for studies targeting bovine mRNA in blood samples, especially when restricted funding source is available.

Animal welfare statement

All the animals handling in accordance with the ethical principles and guidelines adopted by the Brazilian College of Experimentation. All procedures were approved by the Ethical Committee on Animal Experimentation of the Instituto de Zootecnia (Protocol Nr. 298-2020).

Funding

This research study was supported by the São Paulo State Research Support Foundation (FAPESP) (Grant Nr. 2019/22675-6, 2017/11297-5)

Competing Interest/Conflicts of interest

The authors declare that there is no competing interest or conflicts of interest.

Availability of data and material

All experimental data may be available if requested.

Authors' contributions

R.G. and C.H.O.: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Software, Supervision, Validation, Visualization, Writing - original draft, Writing - review & editing; H.N.O, L.M.K., A.E.V.F. and M.C.S.O.: Conceptualization, Supervision, Validation, Visualization, Writing - original draft, Writing - review & editing. All authors read and approved the final manuscript.

Consent to participate

The authors declare that they consent to participate of this manuscript.

Consent for publication

The authors declare that they consent publication of this manuscript.

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How Long Does the mRNA Remains Stable in Untreated Whole Bovine Blood?

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Version 1

Abstract

Figures

Introduction

Material And Methods

Sample collection and storage intervals in common fridge

Extraction of total RNA from whole blood

DNase digestion and cDNA synthesis

Primer design and analysis of the genes

RT-qPCR assay

Statistical analysis

Results

RNA quality and integrity during blood storage at common fridge

Gene regulation

Discussion

Declarations

Animal welfare statement

Funding

Competing Interest/Conflicts of interest

Availability of data and material

Authors' contributions

Consent to participate

Consent for publication

References

Supplementary Files

Archived Versions:

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