Beware of Fixation—It Might Affect Your Experiments

Myoung-Ryoul Park; Karl H. Hasenstein

Open Access

Beware of Fixation—It Might Affect Your Experiments

Myoung-Ryoul Park

and

Karl H. Hasenstein

| Jul 18, 2020

Gravitational and Space Research

Volume 4 (2016): Issue 2 (December 2016)

About this article

Cite

Published Online: Jul 18, 2020

Page range: 47 - 57

DOI: https://doi.org/10.2478/gsr-2016-0012

Keywords
Ethanol, Fixation, Gene Expression, Spaceflight, RNAlater®

© 2020 Myoung-Ryoul Park et al., published by Sciendo

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Seed cassettes used for the germination of Brassica rapa. This hardware was part of the experiment “Magnetophoretic Induction of Curvature in Roots” that flew on Space-X3. The base (bottom) secures the top half of the cassette with peg and nylon screw. Seeds were germinated between ‘germination paper’ (not shown), such that seeds were positioned in one of the grooves. Germination was initiated by adding water through a port (arrow) on the base.

RNA yield of 70% ethanol or RNAlater®-fixed-root or shoot tissue from two-day-old Brassica rapa seedlings that was submersed in fixative gradually (slow fixation), or submersed immediately (fast fixation); details are described in Material and Methods.

Electropherogram of total RNA extracted from two-day-old Brassica rapa tissue after RNAlater® fixative was added gradually (slow fixation), or the tissue was immersed completely (fast fixation).

Electropherogram of total RNA extracted from two-day-old Brassica rapa tissue after 70% ethanol fixative was added gradually (slow fixation), or the tissue was immersed completely (fast fixation).

Comparison of gene transcriptions in Brassica rapa roots after fixation in 70% ethanol or RNAlater®. The relative transcription levels were normalized to slow ethanol fixation of roots (i.e., set to ‘1’) because this normalization resulted in the lowest dynamic range of all possible tissue/fixation combinations.

Overall transcription levels for 70% ethanol and RNAlater®-fixed roots and shoots of two-day-old Brassica rapa seedlings. The values represent the average fold-ratio of examined genes (ACT7, SUS, UBQ1, COX1, Hsp70, and Hsp90) between columns (stress/slow fixation) and rows (immediate, complete submersion in the respective fixative).

EtOH		Slow fixation
EtOH		Root	Shoot
Fast fixation	Root	1.0
	Shoot		1.6
	RNAlater®
	Root	5.0
	Shoot		2.5

Absorbance ratios for RNA extractions of samples extracted by Qiagen RNeasy Plant Mini Kit. The 260/280 ratio indicate RNA purity and the 260/230 value corresponds to protein contamination.

		260/280 ratio		260/230 ratio
	Fixation	Shoot	Root	Shoot	Root
70% EtOH	slow	2.11 ± 0.02	2.10 ± 0.02	1.81 ± 0.02	1.81 ± 0.02
70% EtOH	fast	2.10 ± 0.02	2.11 ± 0.01	1.80 ± 0.07	1.84 ± 0.02
RNAlater®	slow	2.10 ± 0.01	2.13 ± 0.02	1.81 ± 0.04	1.84 ± 0.05
RNAlater®	fast	2.11 ± 0.02	2.07 ± 0.01	1.82 ± 0.03	1.86 ± 0.05

qPCR conditions of gene models, their accession numbers, primers, annealing temperatures, amplicon size, and qPCR efficiency used for the evaluation of slow and fast fixation. Position refers to the relative distance R of the amplicon center from the 3' end of the coding data sequence (R=[d3′-Ac]/CDS); (i.e., 0≡3′, 100≡5′).

Gene (accession #)	Direction	Sequence	® (C)	Amplicon size (bp)*	Efficiency %	Position %CDS

ACT7	Forward	AGCTTCGTGTTGCACCTGAA	52	79	104	73
(AT5G09810.1)	Reverse	ACATGGCAGGGACATTGAAAG	52	79	104	73
SUS	Forward	GTTCAACATTGTCTCTCCTGG	52	53	103	41
(AT5G20830.1)	Reverse	GCTGTAGATGAGCTCCTCGAT	54	53	103	41
UBQ1	Forward	GGAGAGCAGTGACACCATCGA	56	78	104	67
(Z24738.1)	Reverse	GCCAAGGTACGACCATCTTCA	54	78	104	67
COX1	Forward	GTTCCGATTCTGATAGGTGCAC	55	116	103	80
(AY300014.1)	Reverse	CCTACTTCTACTAAGGCTGAGC	55	116	103	80
HSP70	Forward	GTCATCACTGTGCCTGCTTACT	55	101	99	67
(NM_118561.2)	Reverse	CATAAGCCAATGAAGCAGCTGTG	55	101	99	67
HSP90	Forward	AAGGTGACACTGCTAAGCTTGA	53	96	104	82
(AT4G24190.1)	Reverse	CTTCCTTGGTCATACCAATACC	53	96	104	82

eISSN:: 2332-7774
Language:: English

Publication timeframe:: 2 times per year
Journal Subjects:: Life Sciences, other, Materials Sciences, Physics

Journal RSS Feed

Beware of Fixation—It Might Affect Your Experiments

Article Category: Research Article

Published Online: Jul 18, 2020

Page range: 47 - 57

DOI: https://doi.org/10.2478/gsr-2016-0012

Keywords
Ethanol, Fixation, Gene Expression, Spaceflight, RNAlater®

© 2020 Myoung-Ryoul Park et al., published by Sciendo

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Figure 1

Figure 2

Figure 3A

Figure 3B

Figure 4

Absorbance ratios for RNA extractions of samples extracted by Qiagen RNeasy Plant Mini Kit. The 260/280 ratio indicate RNA purity and the 260/230 value corresponds to protein contamination.

Beware of Fixation—It Might Affect Your Experiments

Article Category: Research Article

Published Online: Jul 18, 2020

Page range: 47 - 57

DOI: https://doi.org/10.2478/gsr-2016-0012

KeywordsEthanol, Fixation, Gene Expression, Spaceflight, RNAlater®

© 2020 Myoung-Ryoul Park et al., published by Sciendo

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Figure 1

Figure 2

Figure 3A

Figure 3B

Figure 4

Absorbance ratios for RNA extractions of samples extracted by Qiagen RNeasy Plant Mini Kit. The 260/280 ratio indicate RNA purity and the 260/230 value corresponds to protein contamination.

Keywords
Ethanol, Fixation, Gene Expression, Spaceflight, RNAlater®