Anuran cross-species molecular sensors
Described herein are DNA primer sequences designed for the determination of gene or transcript information from Anuran species, and which may be used in studies for developmental and/or toxicity testing and for environmental toxicology or ecological assessment. Also described herein is a rapid, sensitive, high-throughput assay useful for supporting potential risk assessment across vertebrate clades, and that is also useful for evaluation of complex contaminant mixtures.
1. A method for detecting changes in gene expression of thyroid hormones utilizing a frog tadpole metamorphosis assay method comprising a first phase and a second phase, wherein:
the first phase comprises exposing a tadpole to a compound of interest, removing first and second portions of the tadpole's tail, subjecting the first portion to an apoptosis assay, and
the second phase comprises subjecting the second portion to an assay to detect shifts in a quantity of pre-determined mRNA transcripts, wherein the second phase further comprises subjecting the second portion to qPCR using a primer pair comprising a first primer and a second primer, wherein the primer pair is selected from the group consisting of primer pairs A, D, E, F, G, H, I, J, K, N and O:
Primer
SEQ
SEQ
Pair
First Primer
ID NO:
Second Primer
ID NO:
A
CTGGTGGTGGAT
1
GAGTCTTAAGTCA
2
ATGATGGT
CGGCAAG
D
TGGCTGARGGAT
7
ACAGCAGAGCAGG
8
TYTAYACC
CAAMGA
E
TGATAAGGCCAC
9
CGGGTGATCTTGT
10
AGGRTACCACTA
CGATRA
F
CTCATAGAAGAA
11
GAAGGCTTCTAAG
12
AACAGAGAAAAR
TCCACTTTTCC
AGA
G
CCTGGCTCTSTA
13
RGCTGATCCRAAR
14
YGACTC
TTGAC
H
ASCTCCRCAGAA
15
TCACGTACCAGGC
16
YCAGCA
CAAAA
I
CYGCTCAGTGTC
17
ARGGGCCGGTACT
18
TGGTGT
TGTTT
J
GTTGCCATGARG
19
CCATTGCCWGTGC
20
GWGGAC
AGATA
K
TACAACAGCTGC
21
TCCTTGTCATAGT
22
ACTGAT
CTTCTGT
N
GCTWCAYCCAGA
27
GCTGYTCCTCCTG
28
GTGGTCGTT
CTCCA
O
YATGACYGATGC
29
CATAWCCCTTTGG
30
TGTTCTAG
TGTTGC.
2. The assay method of claim 1 , wherein the first phase further comprises determining whether the compound of interest is thyroid axis active.
3. The assay method of claim 1 , wherein the second phase further comprises subjecting the second portion to qPCR using two or more primer pairs.
4. A method for detecting changes in gene expression of thyroid hormones utilizing a frog tadpole metamorphosis assay method, comprising the steps of:
exposing a tadpole to a compound of interest; and
subjecting a tail portion of said tadpole to an assay to detect shifts in a quantity of pre-determined mRNA transcripts, wherein said assay comprises subjecting the portion to qPCR using a primer pair comprising a first primer and a second primer, wherein the primer pair is selected from the group consisting of primer pairs A, D, E, F, G, H, I, J, K, N and O:
Primer
SEQ
SEQ
Pair
First Primer
ID NO:
Second Primer
ID NO:
A
CTGGTGGTGGAT
1
GAGTCTTAAGTCA
2
ATGATGGT
CGGCAAG
D
TGGCTGARGGAT
7
ACAGCAGAGCAGG
8
TYTAYACC
CAAMGA
E
TGATAAGGCCAC
9
CGGGTGATCTTGT
10
AGGRTACCACTA
CGATRA
F
CTCATAGAAGAA
11
GAAGGCTTCTAAG
12
AACAGAGAAAAR
TCCACTTTTCC
AGA
G
CCTGGCTCTSTA
13
RGCTGATCCRAAR
14
YGACTC
TTGAC
H
ASCTCCRCAGAA
15
TCACGTACCAGGC
16
YCAGCA
CAAAA
I
CYGCTCAGTGTC
17
ARGGGCCGGTACT
18
TGGTGT
TGTTT
J
GTTGCCATGARG
19
CCATTGCCWGTGC
20
GWGGAC
AGATA
K
TACAACAGCTGC
21
TCCTTGTCATAGT
22
ACTGAT
CTTCTGT
N
GCTWCAYCCAGA
27
GCTGYTCCTCCTG
28
GTGGTCGTT
CTCCA
O
YATGACYGATGC
29
CATAWCCCTTTGG
30
TGTTCTAG
TGTTGC.