Complex organisation and structure of the ghrelin antisense strand gene GHRLOS, a candidate non-coding RNA gene

BMC Molecular Biology

Table 1 Designations and sequences of oligonucleotides

Name	Sequence (5'-3')	GHRLOS Exon	Ta (°C)	PCR Cycles
5'2-out-F	GATGGCGATGAATGAACACTG	N/A
5'2-out-R	AATCATCTCAGGAATACCTGGA	2	60	35
5'2-in-F	ATGAATGAACACTGCGTTTGC	N/A
5'2-in-R	AAATGGAAGAGATGAGGCGC	2	61	35
Ex1-cRNA-F	CATACAGTTTGAACATTTATTCGCCTCC	1
Ex1-cRNA-R	CTAATACGACTCACTATAGGGAGA CTCTCTCTAAGTTTAGAAGCGCTCATCTG	1	62	25
3'2OF	GAGAGCGCCTCATCTCTTCC	2
3'2OR	GCGAGCACAGAATTAATACGACTC	N/A	63	35
3'2IF	ATGATTTATTGGAGCTCAAAGC	2
3'2IR	GAATTAATACGACTCACTATAGGT	N/A	57	15
3'4	TACGGAACAGAGGAGAGATGC	4	60	35
3' -RACE-adapter	GCGAGCACAGAATTAATACGACTCACTATAGGTTTTTTTTTTTTVN	N/A		N/A
IPCR-out-F	AAATCCCACCTTTAGTCCCA	4	60	35
IPCR-in-F	CTGCCACCTGAGTGTAGAC	4	60	20
IPCR-ALL-R	CACAGGCTTGGAGACTTCC	I
Pthioate-hex	NNNNsNsN	N/A	30	N/A
Phospho-dT	P- GGCCACGCGTCGACTAGTAC(T)₁₈	N/A	55	N/A
GHRL-Real-RT-LK	CGACTGGAGCACGAGGACACTGAGCCAGAGAGCGCTTCTAAACTTA	N/A	N/A
GHRL-Real-F	GCCCCAGCCGACAAGTG	N/A	60	40
Ito4-F	CATGGAAGTCTCCAAGCCTG	I
Ito4-R	CTGCTCTACTGCCTCAATGTC	4	63	34
GHRLOS-Real-RT-LK	CGACTGGAGCACGAGGACACTGACAATCCTCCCTGAGGTTGATCT	4	N/A
GHRLOS-Real-F	CATTGAGGCAGTAGAGCAGTTGA	4
LK	CGACTGGAGCACGAGGACACTGA	N/A	60	40
Ex4-TaqMan	FAM-TGCCGAATGACCACCTACCCTGACTT- BHQ1	4
18S-Real-F	TTCGGAACTGAGGCCATGAT	N/A
18S-Real-R	CGAACCTCCGACTTTCGTTCT	N/A	60	40
F_CAGE	GGGACTGCCTGTAATAGCAC	I
R_CAGEout	CACGACTGTTGTACAAGCTC	1	60	35
R_CAGEin	GGAGGCGAATAAATGTTCAAACTG	1	61	30
ChiOut-F	TGAAAGCCCAGAAGGAGGA	N/A
ChiOut-R	TCTAAGTTTAGAAGCGCTCATCTG	1	63	35
ChiIn-F	CAGAAGGAGGACGATGTGG	N/A
ChiIn-R	CACGACTGTTGTACAAGCTC	1	62	30
UIS231-F	ACAAGTTCAACGATGTGGTG	N/A
UIS231-R	CAAGTGTGAATAATAACCAAGCCC	N/A	55	40

T7 promoter sequence in PCR primers is underlined. Linker sequence (LK) in the primer GHRLOS-Real-RT-LK and GHRL-Real-RT-LK is shown in bold. In primer 3' -RACE-adapter V denotes an A/G/C residue and N denotes A/G/C. Lowercase letters in primer Pthioate-hex denotes phosphothioate linkages. An uppercase P in primer Phospho-dT indicates that the synthetic oligonucleotide was 5' phosphorylated. Annealing temperatures (Ta) of oligonucleotides employed in PCR is shown. The GHRL or GHRLOS exon location of nucleotides are listed, while oligonucleotides spanning synthetic sequences (adapters and linkers) or genes other than GHRL and GHRLOS are denoted as N/A.

ISSN: 1471-2199