Detail
Article
Online article
FT
Medvik - BMC
  • Something wrong with this record ?

Examining potential confounding factors in gene expression analysis of human saliva and identifying potential housekeeping genes

P. Ostheim, SW. Alemu, A. Tichý, I. Sirak, M. Davidkova, MM. Stastna, G. Kultova, S. Schuele, T. Paunesku, G. Woloschak, SA. Ghandhi, SA. Amundson, M. Haimerl, C. Stroszczynski, M. Port, M. Abend

. 2022 ; 12 (1) : 2312. [pub] 20220210

Language English Country Great Britain

Document type Journal Article, Research Support, Non-U.S. Gov't

Persistent link   https://www.medvik.cz/link/bmc22011031

Isolation of RNA from whole saliva, a non-invasive and easily accessible biofluid that is an attractive alternative to blood for high-throughput biodosimetry of radiological/nuclear victims might be of clinical significance for prediction and diagnosis of disease. In a previous analysis of 12 human samples we identified two challenges to measuring gene expression from total RNA: (1) the fraction of human RNA in whole saliva was low and (2) the bacterial contamination was overwhelming. To overcome these challenges, we performed selective cDNA synthesis for human RNA species only by employing poly(A)+-tail primers followed by qRT-PCR. In the current study, this approach was independently validated on 91 samples from 61 healthy donors. Additionally, we used the ratio of human to bacterial RNA to adjust the input RNA to include equal amounts of human RNA across all samples before cDNA synthesis, which then ensured comparable analysis using the same base human input material. Furthermore, we examined relative levels of ten known housekeeping genes, and assessed inter- and intra-individual differences in 61 salivary RNA isolates, while considering effects of demographical factors (e.g. sex, age), epidemiological factors comprising social habits (e.g. alcohol, cigarette consumption), oral hygiene (e.g. flossing, mouthwash), previous radiological diagnostic procedures (e.g. number of CT-scans) and saliva collection time (circadian periodic). Total human RNA amounts appeared significantly associated with age only (P ≤ 0.02). None of the chosen housekeeping genes showed significant circadian periodicity and either did not associate or were weakly associated with the 24 confounders examined, with one exception, 60% of genes were altered by mouthwash. ATP6, ACTB and B2M represented genes with the highest mean baseline expression (Ct-values ≤ 30) and were detected in all samples. Combining these housekeeping genes for normalization purposes did not decrease inter-individual variance, but increased the robustness. In summary, our work addresses critical confounders and provides important information for the successful examination of gene expression in human whole saliva.

References provided by Crossref.org

000      
00000naa a2200000 a 4500
001      
bmc22011031
003      
CZ-PrNML
005      
20220506131140.0
007      
ta
008      
220425s2022 xxk f 000 0|eng||
009      
AR
024    7_
$a 10.1038/s41598-022-05670-5 $2 doi
035    __
$a (PubMed)35145126
040    __
$a ABA008 $b cze $d ABA008 $e AACR2
041    0_
$a eng
044    __
$a xxk
100    1_
$a Ostheim, P $u Bundeswehr Institute of Radiobiology affiliated to the University of Ulm, Neuherbergstr. 11, 80937, Munich, Germany. patrickostheim@bundeswehr.org
245    10
$a Examining potential confounding factors in gene expression analysis of human saliva and identifying potential housekeeping genes / $c P. Ostheim, SW. Alemu, A. Tichý, I. Sirak, M. Davidkova, MM. Stastna, G. Kultova, S. Schuele, T. Paunesku, G. Woloschak, SA. Ghandhi, SA. Amundson, M. Haimerl, C. Stroszczynski, M. Port, M. Abend
520    9_
$a Isolation of RNA from whole saliva, a non-invasive and easily accessible biofluid that is an attractive alternative to blood for high-throughput biodosimetry of radiological/nuclear victims might be of clinical significance for prediction and diagnosis of disease. In a previous analysis of 12 human samples we identified two challenges to measuring gene expression from total RNA: (1) the fraction of human RNA in whole saliva was low and (2) the bacterial contamination was overwhelming. To overcome these challenges, we performed selective cDNA synthesis for human RNA species only by employing poly(A)+-tail primers followed by qRT-PCR. In the current study, this approach was independently validated on 91 samples from 61 healthy donors. Additionally, we used the ratio of human to bacterial RNA to adjust the input RNA to include equal amounts of human RNA across all samples before cDNA synthesis, which then ensured comparable analysis using the same base human input material. Furthermore, we examined relative levels of ten known housekeeping genes, and assessed inter- and intra-individual differences in 61 salivary RNA isolates, while considering effects of demographical factors (e.g. sex, age), epidemiological factors comprising social habits (e.g. alcohol, cigarette consumption), oral hygiene (e.g. flossing, mouthwash), previous radiological diagnostic procedures (e.g. number of CT-scans) and saliva collection time (circadian periodic). Total human RNA amounts appeared significantly associated with age only (P ≤ 0.02). None of the chosen housekeeping genes showed significant circadian periodicity and either did not associate or were weakly associated with the 24 confounders examined, with one exception, 60% of genes were altered by mouthwash. ATP6, ACTB and B2M represented genes with the highest mean baseline expression (Ct-values ≤ 30) and were detected in all samples. Combining these housekeeping genes for normalization purposes did not decrease inter-individual variance, but increased the robustness. In summary, our work addresses critical confounders and provides important information for the successful examination of gene expression in human whole saliva.
650    _2
$a dospělí $7 D000328
650    _2
$a kontaminace DNA $7 D059018
650    _2
$a komplementární DNA $7 D018076
650    _2
$a ženské pohlaví $7 D005260
650    12
$a exprese genu $7 D015870
650    _2
$a stanovení celkové genové exprese $x metody $7 D020869
650    12
$a esenciální geny $7 D020043
650    _2
$a lidé $7 D006801
650    _2
$a mužské pohlaví $7 D008297
650    _2
$a lidé středního věku $7 D008875
650    _2
$a RNA $x izolace a purifikace $7 D012313
650    _2
$a bakteriální RNA $7 D012329
650    _2
$a kvantitativní polymerázová řetězová reakce $7 D060888
650    _2
$a sliny $x metabolismus $7 D012463
650    _2
$a mladý dospělý $7 D055815
655    _2
$a časopisecké články $7 D016428
655    _2
$a práce podpořená grantem $7 D013485
700    1_
$a Alemu, S W $u Bundeswehr Institute of Radiobiology affiliated to the University of Ulm, Neuherbergstr. 11, 80937, Munich, Germany
700    1_
$a Tichý, A $u Department of Radiobiology, Faculty of Military Health Sciences in Hradec Kralove, University of Defence in Brno, Brno, Czech Republic $u Biomedical Research Centre, University Hospital, Hradec Králové, Czech Republic
700    1_
$a Sirak, I $u Department of Oncology and Radiotherapy, University Hospital and Medical Faculty in Hradec Kralove, Hradec Králové, Czech Republic
700    1_
$a Davidkova, M $u Department of Radiation Dosimetry, Nuclear Physics Institute of the Czech Academy of Sciences, Prague, Czech Republic
700    1_
$a Stastna, M Markova $u Institute for Hematology and Blood Transfusion, Hospital Na Bulovce, Prague, Czech Republic
700    1_
$a Kultova, G $u Department of Radiobiology, Faculty of Military Health Sciences in Hradec Kralove, University of Defence in Brno, Brno, Czech Republic
700    1_
$a Schuele, S $u Bundeswehr Institute of Radiobiology affiliated to the University of Ulm, Neuherbergstr. 11, 80937, Munich, Germany
700    1_
$a Paunesku, T $u Department of Radiation Oncology, Northwestern University, Chicago, IL, 60611, USA
700    1_
$a Woloschak, G $u Department of Radiation Oncology, Northwestern University, Chicago, IL, 60611, USA
700    1_
$a Ghandhi, S A $u Center for Radiological Research, Columbia University Irving Medical Center, New York, NY, 10032, USA
700    1_
$a Amundson, S A $u Center for Radiological Research, Columbia University Irving Medical Center, New York, NY, 10032, USA
700    1_
$a Haimerl, M $u Department of Radiology, University Hospital Regensburg, Regensburg, Germany
700    1_
$a Stroszczynski, C $u Department of Radiology, University Hospital Regensburg, Regensburg, Germany
700    1_
$a Port, M $u Bundeswehr Institute of Radiobiology affiliated to the University of Ulm, Neuherbergstr. 11, 80937, Munich, Germany
700    1_
$a Abend, M $u Bundeswehr Institute of Radiobiology affiliated to the University of Ulm, Neuherbergstr. 11, 80937, Munich, Germany
773    0_
$w MED00182195 $t Scientific reports $x 2045-2322 $g Roč. 12, č. 1 (2022), s. 2312
856    41
$u https://pubmed.ncbi.nlm.nih.gov/35145126 $y Pubmed
910    __
$a ABA008 $b sig $c sign $y p $z 0
990    __
$a 20220425 $b ABA008
991    __
$a 20220506131132 $b ABA008
999    __
$a ok $b bmc $g 1788898 $s 1162229
BAS    __
$a 3
BAS    __
$a PreBMC
BMC    __
$a 2022 $b 12 $c 1 $d 2312 $e 20220210 $i 2045-2322 $m Scientific reports $n Sci Rep $x MED00182195
LZP    __
$a Pubmed-20220425

Find record

Citation metrics

Archiving options