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Time course analysis of RNA stability in human placenta.

Fajardy I, Moitrot E, Vambergue A, Vandersippe-Millot M, Deruelle P, Rousseaux J - BMC Mol. Biol. (2009)

Bottom Line: We tested and compared RNA yields, total RNA integrity, mRNA integrity and stability in these two protocols according to the duration of storage.A long time tissue storage had little effect on the total RNA and mRNA integrity but induced changes in the transcript levels of stress-responsive genes as TNF-alpha or COX2 after 48 h.Rapid tissue handling without dissection and using RNA-stabilizing solution (RNAlater) is a prerequisite to obtain suitable RNA integrity and stability.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centre de Biologie Pathologie, Pôle de Biochimie et Biologie Moléculaire, CHRU de Lille, Université Lille 2, France. i-fajardy@chru-lille.fr

ABSTRACT

Background: Evaluation of RNA quality is essential for gene expression analysis, as the presence of degraded samples may influence the interpretation of expression levels. Particularly, qRT-PCR data can be affected by RNA integrity and stability. To explore systematically how RNA quality affects qRT-PCR assay performance, a set of human placenta RNA samples was generated by two protocols handlings of fresh tissue over a progressive time course of 4 days. Protocol A consists of a direct transfer of tissue into RNA-stabilizing solution (RNAlater) solution. Protocol B uses a dissection of placenta villosities before bio banking. We tested and compared RNA yields, total RNA integrity, mRNA integrity and stability in these two protocols according to the duration of storage.

Results: A long time tissue storage had little effect on the total RNA and mRNA integrity but induced changes in the transcript levels of stress-responsive genes as TNF-alpha or COX2 after 48 h. The loss of the RNA integrity was higher in the placental tissues that underwent a dissection before RNA processing by comparison with those transferred directly into RNA later solution. That loss is moderate, with average RIN (RNA Integration Numbers) range values of 4.5-6.05, in comparison with values of 6.44-7.22 in samples directly transferred to RNAlater (protocol A). Among the house keeping genes tested, the B2M is the most stable.

Conclusion: This study shows that placental samples can be stored at + 4 degrees C up to 48 h before RNA extraction without altering RNA quality. Rapid tissue handling without dissection and using RNA-stabilizing solution (RNAlater) is a prerequisite to obtain suitable RNA integrity and stability.

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Analysis of total RNA integrity according to delay time of storage and to handling conditions. The integrity of total RNA in placental tissues stored at + 4°C for 0 to 96 H, was determined by Agilent Bioanalyzer assay. Results (mean +/- SEM, each experiment in duplicate) were expressed as RIN values (top of the figure), or 28S:18S ratios (bottom of the figure). RNA was prepared from placental samples transferred directly to RNA later™(protocol A), or dissected before banking (protocol B). p values were determined by ANOVA. p < 0.05 was considered to be significant. RIN protocol A versus Protocol B: p = 0.007.
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Figure 2: Analysis of total RNA integrity according to delay time of storage and to handling conditions. The integrity of total RNA in placental tissues stored at + 4°C for 0 to 96 H, was determined by Agilent Bioanalyzer assay. Results (mean +/- SEM, each experiment in duplicate) were expressed as RIN values (top of the figure), or 28S:18S ratios (bottom of the figure). RNA was prepared from placental samples transferred directly to RNA later™(protocol A), or dissected before banking (protocol B). p values were determined by ANOVA. p < 0.05 was considered to be significant. RIN protocol A versus Protocol B: p = 0.007.

Mentions: We evaluated the concentration of each total RNA extract for each delay time according to handling protocol. Results were expressed as RNA yields (μg/mg of tissue). The range varies from 0.14 μg/mg to 0.57 μg/mg. The overall yield was slightly but significantly lower in protocol B (dissection of tissue before transfer) compared to protocol A (direct transfer to RNA later™) at any time (p = 0.01) (Fig. 1). We did not notice any difference of RNA concentration between vaginal and caesarean deliveries (data not shown).


Time course analysis of RNA stability in human placenta.

Fajardy I, Moitrot E, Vambergue A, Vandersippe-Millot M, Deruelle P, Rousseaux J - BMC Mol. Biol. (2009)

Analysis of total RNA integrity according to delay time of storage and to handling conditions. The integrity of total RNA in placental tissues stored at + 4°C for 0 to 96 H, was determined by Agilent Bioanalyzer assay. Results (mean +/- SEM, each experiment in duplicate) were expressed as RIN values (top of the figure), or 28S:18S ratios (bottom of the figure). RNA was prepared from placental samples transferred directly to RNA later™(protocol A), or dissected before banking (protocol B). p values were determined by ANOVA. p < 0.05 was considered to be significant. RIN protocol A versus Protocol B: p = 0.007.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2664811&req=5

Figure 2: Analysis of total RNA integrity according to delay time of storage and to handling conditions. The integrity of total RNA in placental tissues stored at + 4°C for 0 to 96 H, was determined by Agilent Bioanalyzer assay. Results (mean +/- SEM, each experiment in duplicate) were expressed as RIN values (top of the figure), or 28S:18S ratios (bottom of the figure). RNA was prepared from placental samples transferred directly to RNA later™(protocol A), or dissected before banking (protocol B). p values were determined by ANOVA. p < 0.05 was considered to be significant. RIN protocol A versus Protocol B: p = 0.007.
Mentions: We evaluated the concentration of each total RNA extract for each delay time according to handling protocol. Results were expressed as RNA yields (μg/mg of tissue). The range varies from 0.14 μg/mg to 0.57 μg/mg. The overall yield was slightly but significantly lower in protocol B (dissection of tissue before transfer) compared to protocol A (direct transfer to RNA later™) at any time (p = 0.01) (Fig. 1). We did not notice any difference of RNA concentration between vaginal and caesarean deliveries (data not shown).

Bottom Line: We tested and compared RNA yields, total RNA integrity, mRNA integrity and stability in these two protocols according to the duration of storage.A long time tissue storage had little effect on the total RNA and mRNA integrity but induced changes in the transcript levels of stress-responsive genes as TNF-alpha or COX2 after 48 h.Rapid tissue handling without dissection and using RNA-stabilizing solution (RNAlater) is a prerequisite to obtain suitable RNA integrity and stability.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centre de Biologie Pathologie, Pôle de Biochimie et Biologie Moléculaire, CHRU de Lille, Université Lille 2, France. i-fajardy@chru-lille.fr

ABSTRACT

Background: Evaluation of RNA quality is essential for gene expression analysis, as the presence of degraded samples may influence the interpretation of expression levels. Particularly, qRT-PCR data can be affected by RNA integrity and stability. To explore systematically how RNA quality affects qRT-PCR assay performance, a set of human placenta RNA samples was generated by two protocols handlings of fresh tissue over a progressive time course of 4 days. Protocol A consists of a direct transfer of tissue into RNA-stabilizing solution (RNAlater) solution. Protocol B uses a dissection of placenta villosities before bio banking. We tested and compared RNA yields, total RNA integrity, mRNA integrity and stability in these two protocols according to the duration of storage.

Results: A long time tissue storage had little effect on the total RNA and mRNA integrity but induced changes in the transcript levels of stress-responsive genes as TNF-alpha or COX2 after 48 h. The loss of the RNA integrity was higher in the placental tissues that underwent a dissection before RNA processing by comparison with those transferred directly into RNA later solution. That loss is moderate, with average RIN (RNA Integration Numbers) range values of 4.5-6.05, in comparison with values of 6.44-7.22 in samples directly transferred to RNAlater (protocol A). Among the house keeping genes tested, the B2M is the most stable.

Conclusion: This study shows that placental samples can be stored at + 4 degrees C up to 48 h before RNA extraction without altering RNA quality. Rapid tissue handling without dissection and using RNA-stabilizing solution (RNAlater) is a prerequisite to obtain suitable RNA integrity and stability.

Show MeSH