Data in support of qPCR primer design and verification in a Pink1 −/− rat model of Parkinson disease

Abstract

Datasets provided in this article represent the Rattus norvegicus primer design and verification used in Pink1 −/− and wildtype Long Evans brain tissue. Accessible tables include relevant information, accession numbers, sequences, temperatures and product length, describing primer design specific to the transcript amplification use. Additionally, results of Sanger sequencing of qPCR reaction products (FASTA aligned sequences) are presented for genes of interest. Results and further interpretation and discussion can be found in the original research article “Atp13a2 expression in the periaqueductal gray is decreased in the Pink1 −/− rat model of Parkinson disease” [1].

Specifications table

Subject area	Biology
More specific subject area	Neurobiology of disease
Type of data	Tables
How data was acquired	National Center for Biotechnology Information (NCBI) Primer Blast was used to design primers and Sanger sequencing was used for primer confirmation.
Data format	Raw
Experimental factors	Netprimer® (PREMIER Biosoft, Palo Alto, CA, USA) was used to examine secondary structure of all primers designed through NCBI Primer Blast to avoid primer products. Non-template controls were run with each primer pair to check for formation of primer-dimers and non-specific amplification products.
Experimental features	Specificity for each primer pair was confirmed using melt curve analysis; all primer runs yielded single peak melt curves indicating amplification of single gene products. Furthermore, the qPCR reaction product for each gene was sequenced using Sanger sequencing with both forward and reverse primers at the University of Wisconsin Biotechnology Center to confirm that sequences match intended targets.
Data source location	Madison, Wisconsin, USA
Data accessibility	Data are within this article

Value of the data

• •Data presented here allows for experimental replication.
• •Data can be used as a benchmark for other researchers using rat brain tissue.
• •Primers can then be manufactured and used in alternative models of Parkinson disease and then compared to this data set.

1. Data

Table 1 describes the rat (Rattus norvegicus) primer information including gene name, gene abbreviation, GenBank® accession numbers, experimental primer sequences, melt temperature and product length (base pairs) for each gene (Pink1, Asyn, Th, D1, D2, Atp13a2, Gba, Cflar, Gabrb2, Gad1, Gad2) as well as reference genes (Gapdh, βactin).

Table 1.

Rattus norvegicus primer information.

Gene	Gene abbreviation	Accession number	Direction	Sequences	T (°C)	Product (bp)
Gapdh glyceraldehyde-3-phosphate dehydrogenase	Gapdh	NM_017008.4	Forward	GGATACTGAGAGCAAGAGAGA	59	106
			Reverse	TTATGGGGTCTGGGATGGAA
Actb actin, beta	βactin	NM_031144.3	Forward	TGTGGATTGGTGGCTCTATC	59	149
			Reverse	AGAAAGGGTGTAAAACGCAG
Pink1 PTEN induced putative kinase 1	Pink1	Primers created from Dave et al. [2]	Forward	CATGGCTTTGGATGGAGAGT	58	n/a
			Reverse	TGGGAGTTTGCTCTTCAAGG
Snca synuclein, alpha (non-A4 component of amyloid precursor)	Asyn	NM_019169.2	Forward	TCAGCCCAGAGCCTTTCAC	58	165
			Reverse	AGCCACAACTCCCTCCTTG
Th tyrosine hydroxylase	Th	NM_012740.3	Forward	CTTTGACCCAGACACAGCA	59	123
			Reverse	TGGATACGAGAGGCATAGTTC
Drd1 dopamine receptor D1	D1	NM_012546.2	Forward	GCTGGCTCCCTTTCTTCATC	60	111
			Reverse	CACCCAAACCACACAAACAC
Drd2 dopamine receptor D2	D2	NM_012547.1	Forward	TCCTTGACCTTCCTCTTGGG	60	188
			Reverse	CCTGACACTGATGTTGCCTG
Atp13a2 ATPase type 13A2	Atp13a2	NM_001173432.1	Forward	CTTCTCTCTGTCTGGCTTCC	60	95
			Reverse	TCCTCAGTCCGTTGGTGTAG
Gba glucosidase, beta, acid	Gba	NM_001127639.1	Forward	GAGCAGAGTGTTCGGTTAGG	60	115
			Reverse	GATTCAGGGCAAGGTTCCAG
Cflar CASP8 and FADD-like apoptosis regulator	Cflar	NM_001033864.2	Forward	GTGCTGCTGATGGAGATTGG	60	107
			Reverse	CTCTTGTCCTTGGCTACCTTG
Gabrb2 gamma-aminobutyric acid (GABA) A receptor, beta 2	Gabrb2	NM_012957.2	Forward	GGTGCTTTGTCTTTGTCTTTATGG	61	130
			Reverse	CGCATCTTCTCGTTGTTGG
Gad1 glutamate decarboxylase 1	Gad1	NM_017007.1	Forward	GACACTTGAACAGTAGAGACCC	61	116
			Reverse	TGTAGGACGCAGGTTGGTAG
Gad2 glutamate decarboxylase 2	Gad2	NM_012563.1	Forward	CCAGGCTCATCGCATTCAC	61	190
			Reverse	GCACTCACCAGGAAAGGAAC

Table 2 describes the results of Sanger sequencing of qPCR reaction product for each amplification product from the University of Wisconsin Biotechnology Center. Confirmed results presented are FASTA sequences confirmed through NCBI Nucleotide BLAST software.

Table 2.

Results of Sanger sequencing of qPCR reaction product.

Gene	FASTA (Aligned Sequence)
Gapdh	ATCCCAACTCGGCCCCCAACACTGAGCATCTCCCTCACAATTTCCATCCCAGACCCCCATAA
βactin	AGATGTGGATCAGCAAGCAGGAGTACGATGAGTCCGGCCCCTCCATCGTGCACCGCAAATGCTTCTAGGCGGACTGTTAC
PINK1	CTCTTCTCATTTTTCCCGACCAC
Asyn	GGGGAAAACAGGAGGAATCAGAGTTCTGCGGAAGCCTAGAGAGCCGTGTGGAGCAAAGATACATCTTTAGCCATGGATGT
Th	CCAGCCTGTGTACTTTGTGTCCGAGAGCTTCAATGACGCCAAGGACAAGCTCAGG
D1	GGCTCCCTTTCTTCATCTCGAACTGTATGGTGCCCTTCTGTGGCTCTGAGGAGACCCAGCCAT
D2	TTCCTTGACCTTCCTCTTGGGCACAGAAACTAGCTCAGTGGTCGAGCACACCCTGATCGCTGG
Atp13a2	CGGTGTCTAAGGGGGCACCCTTCCGCCAGCCGCTCTACACCAACGGACTGAGGAA
Gba	GCAACTGTTACCACGTCAATTCCATG
Cflar	CTGATGGAGATTGGGGAGAATTTGAATCAATCTGATGTATCCTCCTTAATTT
Gabrb2	TCTTCTTTGGGAGAGGACCCCAGCGCCAAAAGAAAGCAGCTGAGAAAGCTGCTAATGCCAACAACGAGAAGATGCG
Gad1	GCATCTTCCACGCCTTCGCCTGCAACCTCCTCGAACGCGGGAGCGGATCCTAATACTACCAACCTGCGTCCTACAA
Gad2	GCCTTGGGGATCGGAACAGACAGCGTGATTCTGATTAAATGTGATGAGAGAGGGAAAATGATCCCATCTGACCTTGAAAG

2. Experimental design, materials and methods

Netprimer (PREMIER Biosoft, Palo Alto, CA, USA) was used to examine secondary structure of all primers designed through NCBI Primer Blast to avoid primer products (Table 1). The Pink1 gene primer was used based on a previous publication [2]. Non-template controls were run with each primer pair to check for formation of primer-dimers and non-specific amplification products. Specificity for each primer pair was confirmed using melt curve analysis; all primer runs yielded single peak melt curves indicating amplification of single gene products. Furthermore, the qPCR reaction product for each gene was sequenced using Sanger sequencing with both forward and reverse primers at the University of Wisconsin Biotechnology Center (Table 2). FASTA sequences were entered into the NCBI Nucleotide BLAST software to confirm that sequences matched intended targets.

Transparency document. Supplementary material

Supplementary material