lp night blindness.pdf

DOI: 10.1534/genetics.108.088807

Differential Gene Expression of TRPM1 , the Potential Cause of

Congenital Stationary Night Blindness and Coat Spotting

Patterns ( LP ) in the Appaloosa Horse ( Equus caballus )

Rebecca R. Bellone,* ,1 Samantha A. Brooks, † Lynne Sandmeyer, ‡ Barbara A. Murphy, §

George Forsyth,** Sheila Archer, †† Ernest Bailey † and Bruce Grahn ‡

* Department of Biology, University of Tampa, Tampa, Florida 33606, † Department of Veterinary Science, University of Kentucky, Lexington,

Kentucky 40546, ‡ Department of Small Animal Clinical Sciences and ** Department of Biomedical Sciences, Western College of Veterinary

Medicine, University of Saskatchewan, Saskatoon, Saskatchewan S7N5B4, Canada, § School of Agriculture, Food Science and Veterinary

Medicine, University College Dublin, Dublin 4, Ireland and †† Quill Lake, Saskatchewan S0A3E0, Canada

Manuscript received March 4, 2008

Accepted for publication May 8, 2008

ABSTRACT

The appaloosa coat spotting pattern in horses is caused by a single incomplete dominant gene ( LP ).

Homozygosity for LP ( LP/LP ) is directly associated with congenital stationary night blindness (CSNB) in

Appaloosa horses. LP maps to a 6-cM region on ECA1. We investigated the relative expression of two

functional candidate genes located in this LP candidate region ( TRPM1 and OCA2 ), as well as three other

linked loci ( TJP1 , MTMR10 ,and OTUD7A ) by quantitative real-time RT–PCR. No large differences were

found for expression levels of TJP1 , MTMR10 , OTUD7A ,and OCA2 .However, TRPM1 ( Transi ent Rec eptor

Potential Cation Channel , Subfamily M , Member 1 ) expression in the retina of homozygous appaloosa horses was

0.05% the level found in non-appaloosa horses ( R ¼ 0.0005). This constitutes a . 1800-fold change (FC)

decrease in TRPM1 gene expression in the retina (FC ¼ 1870.637, P ¼ 0.001) of CSNB-affected ( LP/LP )

horses. TRPM1 was also downregulated in LP/LP pigmented skin ( R ¼ 0.005, FC ¼ 193.963, P ¼ 0.001)

and in LP/LP unpigmented skin ( R ¼ 0.003, FC ¼ 288.686, P ¼ 0.001) and was downregulated to a lesser

extent in LP/lp unpigmented skin ( R ¼ 0.027, FC ¼ 36.583, P ¼ 0.001). TRP proteins are thought to have a

role in controlling intracellular Ca 2 1 concentration. Decreased expression of TRPM1 in the eye and the skin

may alter bipolar cell signaling as well as melanocyte function, thus causing both CSNB and LP in horses.

C OAT color has been a fascinating topic of genetic

have been reported, including chestnut, frame overo,

cream, black, silver dapple, sabino-1 spotting, tobiano

spotting, and dominant white spotting (Marklund et al.

1996; Metallinos et al. 1998; Mariat et al. 2003; Rieder

et al. 2001; Brooks and Bailey 2005; Brunberg et al.

2006; Brooks et al. 2007; Haase et al. 2007). The

mechanism behind appaloosa spotting, a popular coat

pattern occurring in several breeds of horses, remains to

be elucidated. Likewise, although there are several inher-

ited ocular diseases reported in the horse (cataracts,

glaucoma, anterior segment dysgenesis, and congenital

stationary night blindness), the modes of inheritance,

genetic mutations, and the pathogenesis of these ocular

disorders remain unknown.

Appaloosa spotting is characterized by patches of white

in the coat that tend to be symmetrical and centered over

the hips. In addition to the patterning in the coat,

appaloosa spotted horses have three additional pigmen-

tation traits: striped hooves, readily visible nonpigmented

sclera around the eye, and mottled pigmentation around

the anus, genitalia, and muzzle (Sponenberg and Beaver

1983). The extent of spotting varies widely among indi-

viduals, resulting in a collection of patterns that are

termed ‘‘the leopard complex’’ (Sponenberg et al. 1990).

discussion and discovery for over a century. The

pigment genes of mice were one of the ﬁrst genetic

systems to be explored through breeding and transgenic

studies. To date, at least 127 loci involved in pigmen-

tation have been described (Silvers 1979; Bennett and

Lamoreux 2003). The genes that affect pigmentation in

the skin and hair inﬂuence other body systems, and

many of these genes have been studied in different

mammals. One of the most extensively studied examples

is oculocutaneous albinism type 1, a developmental

disorder in humans that affects pigmentation in the skin

and hair, as well as eye development. This disease is

caused by mutations in the tyrosinase gene ( TYR ), which

is involved in the ﬁrst step of melanin production

(Toyofuko et al. 2001; Ray et al. 2007).

Horses ( Equus caballus ) are valued by breeders and

enthusiasts for their beauty and variety of coat color and

patterns. The genetic mechanisms involved in several

different variations of coloration and patterning in horses

1 Corresponding author: Department of Biology, University of Tampa, 401

W. Kennedy Blvd., Box 3F Tampa, FL 33606.

E-mail: rbellone@ut.edu

Genetics 179: 1861–1870 (August 2008)

1862

R. R. Bellone et al.

Figure 1.—Horses dis-

playing different Appaloosa

coat color patterns. (a)

Lace blanket ( LP/lp ). (b)

Spotted blanket ( LP/lp ).

Snowcap blanket ( LP/LP ).

(e) Fewspot ( LP/LP ).

The spectrum of patterns with the leopard complex in-

cludes very minimal white patches on the rump (known

as a ‘‘lace blanket’’), a white body with many oval or round

pigmented spots dispersed throughout (known as ‘‘leop-

ard,’’ from which the genetic locus is named), and nearly

complete depigmentation (known as ‘‘fewspot’’) (Figure

1). A single autosomal dominant gene, leopard complex

( LP ), is thought to be responsible for the inheritance of

these patterns and associated traits, while modiﬁer genes

are thought to play a role in determining the amount

of white patterning that is inherited (Miller 1965;

Sponenberg et al. 1990; S. Archer and R. R. Bellone,

unpublished data). Horses that are homozygous for ap-

paloosa spotting ( LP/LP ) tend to have fewer spots than

heterozygotes on the white patterned areas; these horses

are known as ‘‘fewspots’’ (largely white body with little to

no spots) and ‘‘snowcaps’’ (white over the croup and hips

with little to no spots) (Sponenberg et al. 1990; Lapp and

Carr 1998; Figure 1).

We have recently reported an association between

homozygosity for LP and congenital stationary night

blindness (CSNB) (Sandmeyer et al. 2007). CSNB is

characterized by a congenital and nonprogressive sco-

topic visual deﬁcit (Witzel et al. 1977, 1978; Rebhun

et al. 1984). Affected horses may exhibit apprehension

in dimly lit conditions and may be difﬁcult to train and

handle in phototopic (light) and scotopic (dark) con-

ditions (Witzel et al. 1977, 1978; Rebhun et al. 1984).

Affected animals occasionally manifest a bilateral dor-

somedial strabismus (improper eye alignment) and

nystagmus (involuntary eye movement) (Rebhun et al .

1984; Sandmeyer et al . 2007). CSNB is diagnosed by an

absent b-wave and a depolarizing a-wave in scotopic

(dark-adapted) electroretinography (ERG) (Figure 2).

This ERG pattern is known as a ‘‘negative ERG’’ (Witzel

et al. 1977). No morphological or ultrastructural abnor-

malities have been detected in the retinas of horses with

CSNB (Witzel et al. 1977; Sandmeyer et al. 2007).

A similar ‘‘negative ERG’’ is seen in the Schubert–

Bornshein type of human CSNB (Schubert and Born-

shein 1952; Witzel et al. 1978). This type of CSNB is

thought to be caused by a defective neural transmission

within the retinal rod pathway (Witzel et al . 1977, 1978;

Sandmeyer et al. 2007). Neural transmission is complex

and the mechanism of the transmission defect in CSNB

is not reported. Rod photoreceptors are most sensitive

under scotopic conditions. In the dark, these cells exist

in a depolarized state. They hyperpolarize in response

to light, and signaling occurs through reductions in

glutamate release (Stryer 1991). This hyperpolar-

ization is responsible for the a-wave of the electro-

retinogram. Normally this results in stimulation of a

population of bipolar cells, the ON bipolar cells. The

glutamate receptor of the ON bipolar cells is a metab-

otropic glutamate receptor (MGluR6) and this receptor

is expressed only in the retinal bipolar cell layer

(Nomura et al. 1994; Nakanishi et al. 1998). The MGluR6

receptors sense the reduction in synaptic glutamate and

produce a response that depolarizes the ON bipolar cell

(Nakanishi et al. 1998). This depolarization is respon-

sible for the b-wave of the electroretinogram. The ERG

characteristics of the Schubert–Bornshein type of CSNB

are consistent with a failure in depolarization of the ON

bipolar cell (Sandmeyer et al. 2007).

TRPM1 , a Potential Cause of CSNB and LP in Appaloosas

1863

Figure 2.—Scotopic elec-

troretinogram from an lp/lp

Appaloosa (left) and an LP/

LP Appaloosa with CSNB

(right). Note the absence

of a b-wave in the ERG trac-

ing from the LP/LP horse.

(50 msec, 100 mV).

A whole-genome scanning panel of microsatellite

markers was used to map LP to a 6-cM region on ECA1

(Terry et al. 2004). Prior to the sequencing of the

equine genome, two candidate genes— Transient Re-

ceptor Potential Cation Channel , Subfamily M , Member 1

( TRPM1 ) and Oculoctaneous Albinism Type II ( OCA2 )—

were suggested on the basis of comparative phenotypes

in humans and mice (Terry et al. 2004). Both TRPM1

and OCA2 were FISH mapped to ECA1, to the same

interval as LP (Bellone et al. 2006a). One SNP in the

equine OCA2 gene has been ruled out as the cause for

appaloosa spotting (Bellone et al. 2006b).

TRPM1 ,alsoknownas Melastatin 1 ( MLSN1 ), is a

member of the transient receptor potential (TRP) chan-

nel family. Channels in the TRP family may permit Ca 2 1

entry into hyperpolarized cells, producing intracellular

responses linked to the phosphatidylinositol and protein

kinase C signal transduction pathways (Clapham et al.

2001). TRPs are important in cellular and somatosensory

perception (Nilius 2007). Defects in a light-gaited TRP

channel results in a loss of phototransduction in Dro-

sophila (reviewed in Kim 2004). Although the speciﬁc

function of TRPM1 has yet to be described, cellular

sensation and intercellular signaling are vital for normal

melanocyte migration (reviewed in Steingr´msson et al.

2006). In mice and humans, the promoter region of this

gene contains four consensus binding sites for a melano-

cyte transcription factor, MITF (Hunter et al. 1998; Zhiqi

et al. 2004). One of these sites, termed an M-box, is unique

to melanocytic expression (Hunter et al. 1998). TRPM1 is

downregulated in highly metastatic melanoma cells,

suggesting that this protein plays an important role in

normal melanogenesis (Duncan et al. 1998).

Mutations in the OCA2 gene (also P , or pink-eyed

dilution) cause hypopigmentation phenotypes in mice

(Gardner et al. 1992). Similarly, in humans, mutations

in OCA2 cause the most common form of albinism (Lee

et al. 1994). Additionally, other mutations in this gene

are thought to be responsible for the variation in human

eye color (Duffy et al. 2007; Eiberg et al. 2008). It is

believed that during melanogenesis this protein func-

tions to control intramelanasomal pH and aids in

tryosinase processing (Sturm et al. 2001; Ni-Komatsu

and Orlow 2006).

The objectives of this investigation included determin-

ing if differential gene expression could be the cause of

LP and CSNB. We have evaluated the relative expression

of candidate genes by quantitative real-time RT–PCR. We

further investigated whether a local regulatory phenom-

enon exists by measuring the expression of three

additional nearby genes. These included two genes

positioned on either side of TRPM1 —the OTU domain

containing 7A ( OTUD7A ) and the myotubularin-related

protein 10 ( MTMR10 )—and one gene more distal—

tight junction protein 1 ( TJP1 )—according to the ﬁrst

assembly of the equine genome (http://www.genome.

ucsc.edu/cgi-bin/hgGateway?org ¼ Horse& ¼ equCab1)

(Figure 3).

MATERIALS AND METHODS

Horses and genotype categories: Horses were categorized

according to genotype and phenotype for LP , which was

diagnosed by coat color assessment, breeding records, and,

for those horses used in the retinal study, also by ocular

examination, including scotopic ERG. Horses were included

in the LP/LP group if they had a ‘‘fewspot’’ or ‘‘snowcap

blanket’’ pattern and a scotopic ERG consistent with CSNB

(Figure 1a). Horses in the LP/lp group all displayed white

patterning with dark spots and/or had breeding records

consistent with heterozygosity (‘‘leopard,’’ ‘‘spotted blanket,’’

or ‘‘lace blanket’’ patterns) and a normal scotopic ERG. Horses

were included in the non-appaloosa ( lp/lp ) group if they were

solid colored and showed no other traits associated with the

presence of LP (striped hooves, white sclera, and mottled skin)

and a normal scotopic ERG. The non-appaloosa horses were

from the Thoroughbred and American Quarter Horse breeds,

two breeds that are not known to possess any appaloosa

spotted individuals. Due to the invasive nature of some of the

experiments performed, it was impossible to obtain a signif-

icant number of samples from age, sex, and base-coat-color-

matched horses. Both male and female horses were used in

this study, horses ranged in age from , 1 year to 23 years old,

and the base coat colors of black, bay, and chestnut were all

represented (Table 1).

Figure 3.—Genomic map highlighting those genes tested

for differential expression within LP candidate region.

1864

R. R. Bellone et al.

TABLE 1

Base coat color, proposed LP genotype, disease status, age, sex, and tissue sampled for each horse used in qRT–PCR experiments

Horse sample no.

Base color

Proposed LP genotype

CSNB phenotype

Age at sampling

Sex

Tissue sampled

05-10

Bay dun

LP/LP

CSNB

Mare

Skin

05-12

Black

LP/LP

CSNB

Mare

Skin

05-13

Chestnut

LP/LP

CSNB

Mare

Skin

06-261

Black

LP/LP

Not examined

Stallion

Skin

06-222

Bay

LP/LP

CSNB

5 mo

Mare

Skin

07-51

Liver chestnut

LP/LP

CSNB

Gelding

Skin/retina

07-54

Chestnut

LP/LP

CSNB

Stallion

Skin/retina

07-53

Chestnut

LP/LP

CSNB

Stallion

Retina

07-52

Chestnut

LP/LP

CSNB

Stallion

Retina

05-14

Black

LP/lp

Normal

Stallion

Skin

05-15

Dark bay

LP/lp

Not examined

Stallion

Skin

LP/lp

05-18

Bay dun

Normal

Gelding

Skin

07-49

Chestnut

LP/lp

Normal

Unknown

Gelding

Skin/retina

07-50

Bay

LP/lp

Normal

Gelding

Skin/retina

06-275

Chestnut

LP/lp

Not examined

Mare

Skin

06-268

Black

LP/lp

Normal

Gelding

Skin/retina

06-269

Bay dun

LP/lp

Normal

Gelding

retina

05-48

Red dun

lp/lp

Not examined

Gelding

Skin

05-49

Dark bay

lp/lp

Not examined

Mare

Skin

D052

Bay

lp/lp

Not examined

Stallion

Skin

06-270

Chestnut

lp/lp

Normal

6 mo

Stallion

Skin

06-271

Dark bay

lp/lp

Normal

Mare

Skin/retina

lp/lp

07-46

Chestnut

Normal

Stallion

Skin/retina

07-48

Bay

lp/lp

Normal

Mare

Skin/retina

07-47

Buckskin

lp/lp

Normal

Mare

Retina

07-44

Bay

lp/lp

Normal

Mare

Retina

07-45

Chestnut

lp/lp

Normal

Stallion

Retina

CSNB (Witzel et al. 1977; Sandmeyer et al . 2007). Horses

included in the LP/LP ( n ¼ 4) group had a ‘‘negative ERG,’’

and those in the LP/lp group ( n ¼ 4) and lp/lp group ( n ¼ 6)

had

Ophthalmic examinations: Horses used in this study were

categorized by ocular examination, which included. neuro-

phthalmic examination, slit-lamp biomicroscopy (SL-14,

Kowa, Japan), indirect ophthalmoscopy (Heine Omega 200,

Heine Instruments), and electroretinography (Cadwell Sierra

II, Cadwell Laboratories, Kenewick, WA). For electroretinog-

raphy, horses were sedated with 10 mg/kg detomidine hydro-

chloride (Dormosedan, Orion Pharma, Pﬁzer Animal Health,

Kirkland, QC, Canada) by intravenous bolus. Pharmacological

mydriasis was achieved with 0.2 ml 1% tropicamide (1%

mydriacyl, Alcon, Mississauga, ON, Canada). Auriculopalpe-

bral nerve blocks were performed using 2 ml of a 2% lidocaine

hydrochloride injectable solution (Bimeda-MTC Animal

Health, Cambridge, ON, Canada). Scotopic ERGs were com-

pleted bilaterally to identify nyctalopia and CSNB. A corneal

DTL microﬁber electrode (DTL Plus Electrode, Diagnosys,

Littleton, MA) was placed on the cornea, and platinum

subdermal needle electrodes (Cadwell Low Proﬁle Needle

electrodes, Cadwell Laboratories) were used as reference and

ground. The reference electrode was placed subdermally 3 cm

from the lateral canthus and the ground electrode was placed

subdermally over the occipital bone. The ERGs were elicited

with a white xenon strobe light and recorded with a Cadwell

Sierra II (Cadwell Laboratories) with the bandwidth set at 0.3–

500 Hz; eyelids were held open manually for each test and a

pseudo-Ganzfeld was used to attempt even stimulation of the

entire retina (Komar ´ my et al. 2003). Horses were dark

adapted for 25 min and dark-adapted ERG responses were

stimulated using maximum light intensity with each recording

representing the average of 20 responses. An a-wave domi-

nated ERG or ‘‘negative ERG’’ was considered diagnostic of

normal

scotopic

and

phototopic

electroretinograms

(Figure 2, Table 2).

Retina and collection and RNA isolation: Horses were

humanely euthanized by intravenous overdose of barbiturate

(Euthanyl, MTC Pharmaceuticals) following the Canadian

Council on Animal Care Guidelines for Experimental Animal

Use and approved by the University of Saskatchewan Animal

Care Committee. The eyes were removed immediately and

placed on ice. The posterior segment of the globes were

isolated by removing the anterior segment via a 360 incision

posterior to the limbus. The vitreous was removed by gentle

traction. In one eye from each horse, the retina was detached

from the periphery and was transected at the optic nerve with

Vannas scissors. For the second eye from each horse, the

posterior segment was transected with a scalpel blade and one-

half was prepared for histology. The retina was removed from

TABLE 2

Scotopic ERG results for sample horses used in retinal study

LP/LP

LP/lp

lp/lp

Number

Normal scotopic ERG

‘‘Negative’’ scotopic ERG

TRPM1 , a Potential Cause of CSNB and LP in Appaloosas

1865

TABLE 3

Primer and probe sequences and PCR efﬁciency used in quantitative real-time RT–PCR

Gene

Primer/probe

Sequence

Exon no.

PCR efﬁciency

R 2

b -actin

Forward

5 9 -GCCGTCTTCCCCTCCAT-3 9

2.07

Reverse

5 9 -GCCCACGTATGAGTCCTTCTG-3 9

Probe

5 9 -GGCACCAGGGCGTGATGGTGGGC-3 9

2 and 3

TRPM1

Forward

5 9 -GACGACATCTCCCAGGATCT-3 9

2.09

0.99

Reverse

5 9 -TGCTCGTCGTGCTTATAGGA-3 9

Probe

5 9 -ATTCAAAAGACTTTGGCCAGCTGGC-3 9

16 and 17

OCA2

Forward

5 9 -AGATCAAGGAAAGTTCTGGCAGT-3 9

2.19

0.99

Reverse

5 9 -CTGGAGCAGCGTGGAATC-3 9

Probe

5 9 -AAGCTACTCTGTGAACCTCAGCAGCCAT-3 9

6 and 7

TJP1

Forward

5 9 -ATATGGGAACAACACACAGTGA-3 9

2.18

0.98

Reverse

5 9 -GGTCCTCCTTTCAGCACATC-3 9

Probe

5 9 -CTTCACAGGGCTCCTGGATTTGGAT-3 9

2 and 3

MTMR10

Forward

5 9 -TGTCAGATTTCGCTTTGATGA-3 9

2.28

0.98

Reverse

5 9 -GGTCTGTTGGCTGGGAATAA-3 9

Probe

5 9 -TCAGGTCCTGAAAGTGCCAAAAAGG-3 9

5 and 6

OTUD7A

Forward

5 9 -CAGACTTTGTTCGGTCCACA-3 9

2.27

0.98

Reverse

5 9 -AGTCACTCAGAGCGGCTGTC-3 9

Probe

5 9 -AGAACCTGGTCTGGCCAGAGACCTG-3 9

the remaining posterior segment and added to the entire

retina of the ﬁrst eye. Retina was then centrifuged and

suspended in the appropriate volume of Trizol (Invitrogen)

and homogenized in a Polytron mechanical homogenizer

(Brinkman Instruments, Westbury, NY). Total retinal RNA was

isolated according to the manufacturer’s instructions and

stored at 80 until used.

Skin collection and RNA isolation: Skin samples from seven

homozygous appaloosa spotted horses ( LP/LP ), seven hetero-

zygotes ( LP/lp ), and seven non-appaloosa ( lp/lp ) were ob-

tained. Samples were taken from live horses (with appropriate

consent of owner) and from those euthanized as described

above. Donor skin sites of the live horses were inﬁltrated with a

local anesthetic (2% lidocaine hydrochloride, Bimeda-MTC

Animal Health, Cambridge, ON, Canada). Following hair

removal by shaving the sample area, ﬁve 6-mm dermal punch

biopsies were collected and immediately snap frozen in liquid

nitrogen. Samples were placed at 80 until processing. From

each horse in the LP/LP group and LP/lp group, two sample

areas were collected for RNA extraction: one sample area that

was pigmented ( i.e. , a darkly pigmented body spot) and one

area where skin and hair where completely unpigmented. Skin

samples from euthanized horses were collected in a similar

fashion; however, punch biopsies were not used. Instead 10 3

1-cm 2 sections of skin were harvested from each site by sharp

incision with a sterile no. 22 scalpel blade (Paragon, Shefﬁeld,

England). A new scalpel blade and a new pair of sterile gloves

were worn to perform the harvest from each site to avoid

transfer of genetic material. Prior to RNA isolation, skin

samples were ﬁrst powdered by crushing under liquid nitro-

gen. Total RNA was isolated from 0.5 g of tissue in a buffer of

4 m guanidinium isothiocyanate, 0.1 m Tris–HCl, 25 mm

EDTA (pH 7.5), and 1% (v/v) 2-mercaptoethanol, followed

by differential alcohol and salt precipitations (Chomczynski

and Sacchi 1987; MacLeod et al. 1996). All samples were

stored at 80.

Quantitative real-time RT–PCR: RNA was quantiﬁed using

a NanoDrop spectrophotometer (NanoDrop Technologies,

Wilmington, DE) and sample concentrations were adjusted to

50 ng/ml with RNAse free water (Ambion, Austin, TX). RNA

integrity and purity was veriﬁed using a Bioanalyzer (Agilent

Technologies, Santa Clara, CA). All skin and retinal samples

isolated were of high purity and integrity, and all samples used

had RNA integrity numbers . 8.

Equine homologs for TRPM1 , OCA2 , TJP1 , MTMR10 , and

OTUD7A were identiﬁed from the Entrez Trace Archive using a

Discontiguous Megablast (http://www.ncbi.nih.gov/BLAST)

or by a BLAT search against the horse ( January 2007)

(equCab1) assembly (http://www.genome.ucsc.edu/). Taq-

man primers and probes were designed as previously de-

scribed (Murphy et al. 2006). Preliminary experiments

revealed that b -actin was the most stable reference gene among

those tested in our samples. The PCR efﬁciency of primer/

probe combinations were calculated using serial dilutions of

RNA spanning a magnitude of eightfold (or greater) by the

REST analysis program (Pfafﬂ et al. 2002). R 2 values for

standard curves were $0.98 for all products tested (Table 3).

All primer pairs were tested to ensure that genomic DNA was

not being ampliﬁed by using a minus reverse transcription

control in each assay.

Taqman quantitative real-time RT–PCR was performed

using a Smart Cycler real-time thermal cycler (Cepheid,

Sunnyvale, CA). Each 25-ml reaction contained 250 ng of RNA,

1 3 EZ buffer (Applied Biosystems, Foster City, CA), 300 mm of

each dNTP, 2.5 mm manganese acetate, 200 nm forward and

reverse primer, 125 nm ﬂuorogenic probe, 40 units RNasin

(Roche, Indianapolis), and 2.5 units rTth (Applied Biosystems).

Cepheid also recommends the addition of an ‘‘additive reagent’’

to prevent binding of polymerases and nucleic acids to the

reaction tubes. This reagent was added to give a ﬁnal concentra-

tion of 0.2 mg/ml bovine serum albumin (nonacetylated), 0.15 m

trehalose, and 0.2% Tween 20. Thermocycler parameters for all

assays consisted of a 30-min reverse transcription (RT) step at 60,

2minat94 , and 45 cycles of 94 for 15 sec (denaturation) and

60 for 30 sec (annealing and extension). The threshold crossing

cycle ( C t ) values generated by the Smart Cycler were used to

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