ATRC Reagent Bank
Primer Panel
Human, Rat, and Mouse Targets for Q-PCR
|
TARGET mRNA |
MARKER |
PRIMER SEQUENCE |
Tm |
PRODUCT SIZE (bp) (Linked to Detail) |
DESIGNED BY |
|||
|
Human |
Rat |
Mouse |
||||||
|
β-Actin |
Housekeeping |
F |
ACTCTTCCAGCCTTCCTTC |
56 |
Teresa Hsi |
|||
|
R |
ATCTCCTTCTGCATCCTGTC |
57 |
||||||
|
α-Synuclein |
Neurons |
F |
AGTGACAAATGTTGGAGGAG |
55 |
Ippolita Cantuti-Castelvetri, PhD |
|||
|
R |
GCTTCAGGTTCGTAGTCTTG |
55 |
||||||
|
GAPDH |
Housekeeping |
F |
ATGACATCAAGAAGGTGGTG |
56 |
Teresa Hsi |
|||
|
R |
CATACCAGGAAATGAGCTTG |
56 |
||||||
|
GFAP |
Astrocytes |
F |
TCCTGGAACAGCAAAACAAG |
59 |
Charles Vanderburg, PhD |
|||
|
R |
CAGCCTCAGGTTGGTTTCAT |
60 |
||||||
|
NSE |
Neurons |
F |
TCTGCTGCTCAAGGTCAACC |
62 |
Ippolita Cantuti-Castelvetri, PhD |
|||
|
R |
CGGCACGGGGCACCAGTCTT |
72 |
||||||
|
MBP |
Oligodendrocytes |
F |
CTATAAATCGGCTCACAAGG |
55 |
Teresa Hsi |
|||
|
R |
AGGCGGTTATATTAAGAAGC |
53 |
||||||
|
18S Ribosomal |
Housekeeping |
F |
GTCTGTGATGCCCTTAGATG |
56 |
Teresa Hsi |
|||
|
R |
AGCTTATGACCCGCACTTAC |
57 |
||||||
|
Synaptophysin |
Neurons |
F |
TGCAGAACAAGTACCGAGAG |
57 |
Teresa Hsi |
|||
|
R |
CTGTCTCCTTAAACACGAACC |
56 |
||||||
|
β-Tubulin |
Housekeeping |
F |
TCGTGGAATGGATCCCCAAC |
65 |
Charles Vanderburg, PhD |
|||
|
R |
CTCCATCTCGTCCATGCCCT |
65 |
||||||
|
CNP-1 |
Oligodendrocytes |
F |
TACTTCGGCTGGTTCCTGAC |
60 |
Charles Vanderburg, PhD |
|||
|
R |
GCCTTCCCGTAGTCACAAAA |
59 |
||||||
|
MAC-1 |
Microglia |
F |
CCTTGTTCTCTTTGATGCAG |
56 |
Charles Vanderburg, PhD |
|||
|
R |
GTGATGACAACTAGGATCTT |
49 |
||||||
|
TLR-4 |
Microglia |
F |
GTAAAGAATTTAGAAGAAGG |
46 |
Teresa Hsi |
|||
|
R |
TGAGCAATCTCATATTCAAAG |
53 |
||||||
|
NEFH |
Motor Neuron |
F |
AGTGGTTCCGAGTGAGGTTG |
|
Rachel Diamond |
|||
|
R |
CTGCTGAATAGCGTCCTGGT |
|
||||||
|
NEFM |
Motor Neurons |
F |
AGTGGTTCAAATGCCGCTAC |
|
Rachel Diamond |
|||
|
R |
TTTTCCAACTGCTGGATGGT |
|
||||||
|
NEFL |
Motor Neurons |
F |
CCATGCAGGACACAATCAAC |
|
Rachel Diamond |
|||
|
R |
CGCCTTCCAAGAGTTTTCTG |
|
||||||
|
ChAT |
Motor Neurons |
F |
TCATTAATTTCCGCCGTCTC |
|
Charles Vanderburg, PhD |
|||
|
R |
AGTCCCGGTTGGTGGAGTC |
|
||||||
Method
Primer Design
We designed primer pairs to bind and amplify twelve general and cell-specific markers in human, rat, and mouse tissue. Primers were generated using the Primer3 software (Whitehead Institute for Biomedical Research) and ordered from Invitrogen. In some cases, the regions recognized by the primers are not completely homologous between the three species. When such discrepancies occur, primers were designed using the human sequence, except in the case of β-tubulin, where the mouse sequence was used.
cDNA Preparation
For PCR testing, we made ten-fold serial dilutions of normal human, wtSD-rat, and wtC57-mouse brain cDNAs (available in the ATRC Reagent Bank) in nuclease-free water. 2 microliters of template were used in each reaction.
Real-Time PCR
Primers were tested for specificity in all three species by performing real-time PCR on a Bio-Rad iQTM Real-Time PCR Detection System. The following per-well reaction mix was prepared:
- 12.5 μL of iQTM 2X SYBR Green® Supermix (Bio-Rad)
- 1 μL of 10 μM forward primer (final concentration 400nM)
- 1 μL of 10 μM reverse primer (final concentration 400nM)
- 2 μL of cDNA
- 8.5 of μL nuclease-free water
PCR conditions were 6 minutes at 95°C followed by 40 cycles of 30 seconds at 95°C, 30 seconds at 55°C, and 45 seconds at 72°C. Fluorescence data collection was enabled during the 72°C step, above the melting temperature of nonspecific products (e.g. primer-dimers) and was followed by a melt curve analysis.
Product Verification
PCR products were verified both by the presence of a melt curve peak representing a specific product and by detection of a single band of the expected size in the Agilent 2100 Bioanalyzer. Products were sequenced at the MGH DNA Core facility. NCBI’s BLAST software confirmed homology of our products with the target genes.
Brain RNAs
Method
RNA Preparation
We prepared total RNA from homogenates of normal human, wild-type Sprague-Dawley rat, and wild-type C57 mouse brain tissue using Tri-reagent (Sigma). Our extraction protocol can be found in the ATRC’s Online Document Repository. RNA was eluted in nuclease-free water and its quality was verified on the Agilent 2100 Bioanalyzer (below). Concentration and purity were tested on a UV spectrophotometer.
Normal Human Brain: Frontal Cortex (ADRC 1048)
Concentration: 606.87 ng/μL A260/280 1.71
Sprague-Dawley Rat: Whole Brain
Concentration: 5532.4 ng/μL A260/280 1.71
C57 Mouse: Whole Brain
Concentration: 4602.3 ng/μL A260/280 1.67
In addition to our normal brain RNAs, the ATRC has several other normal and disease-state human brain RNAs immediately available. Quantities are limited, and the samples on this list are expected to change frequently. If you are looking for a brain RNA-type that is not listed here, feel free to contact us for assistance in locating a sample and testing RNA quality.
Alzheimer’s Human Brain: Frontal Cortex (ADRC 1076)
ALS Human Brain: Frontal Cortex (ADRC 1134)
Alzheimer’s Human Brain: Cerebellum (ADRC 1183)
Normal Human Brain: Frontal Cortex (ADRC 1188)
Normal Human Brain: Cerebellum (ADRC 1188)
Alzheimer’s Human Brain: Frontal Cortex (ADRC 1202)
Alzheimer’s Human Brain: Cerebellum (ADRC 1202)
Leukodystrophy Human Brain: Cerebellum (ADRC 1203)
Parkinson’s Human Brain: Cerebellum (ADRC 1229)
Hungtington’s Human Brain: Cerebellum (ADRC 1246)
Huntington’s Human Brain: Thalamus (ADRC 1246)
Huntington’s Human Brain: Cerebellum (ADRC 1247)
Brain cDNAs
Method
cDNA Preparation
First-strand cDNAs were synthesized using 5μg RNA from normal human, wtSD-rat, and wtC57-mouse brain tissue. We used SupersScriptTM II Reverse Transcriptase (Invitrogen) according to the manufacturer’s instructions, priming with random hexamers.