PCR run on samples from adult mosquitoes reared in cages contaminated with densonucleosis virus using primers specific for the virion proteins with an expected size of 372 bp. Lane 1 is the negative control. Lanes 2 to 4 are the negative control cage samples. Lanes 5 to 9 and 12 to 19 are the infected cage samples. Lane 10 is the positive control. Lane 11 is the 100 bp ladder with 100 bp at the bottom; the heavy band at the top is 500 bp. Note that all 13 infected cage samples run were virion protein positive. (Erica Suchman, Colorado State University, Ft. Collins, CO)

PCR run on samples from adult mosquitoes reared in cages contaminated with densonucleosis virus using primers specific for the virion proteins with an expected size of 372 bp. Lane 1 is the negative control. Lanes 2 to 4 are the negative control cage samples. Lanes 5 to 9 and 12 to 19 are the infected cage samples. Lane 10 is the positive control. Lane 11 is the 100 bp ladder with 100 bp at the bottom; the heavy band at the top is 500 bp.  Note that all 13 infected cage samples run were virion protein positive. (Labeled view) (Erica Suchman, Colorado State University, Ft. Collins, CO)

PCR run on samples from adult mosquitoes reared in cages contaminated with densonucleosis virus using primers specific for the virion proteins with an expected size of 372 bp.  The initial samples were  taken 0 to 24 hours post adult emergence in contaminated cages. Lane 1 is the negative control. Lanes 2 to 4 are samples from the control cage. Lanes 5 to 8 and 11 to 19 are samples from infected cages. Lane 9 is the positive control.  Lane 10 is the 100 bp ladder with 100 bp at the bottom; the heavy band at the top is 500 bp.  Note that all samples are negative. (Erica Suchman, Colorado State University, Ft. Collins, CO)

PCR run on samples from adult mosquitoes reared in cages contaminated with densonucleosis virus using primers specific for the virion proteins with an expected size of 372 bp.  Initial samples were taken 0 to 24 hours post adult emergence in contaminated cages. Lane 1 is the negative control.  Lanes 2 to 4 are samples from the control cage. Lanes 5 to 8 and 11 to 19 are samples from infected cages. Lane 9 is the positive control.  Lane 10 is the 100 bp ladder with 100 bp at the bottom; the heavy band at the top is 500 bp. Note that all samples are negative. (Erica Suchman, Colorado State University, Ft. Collins, CO)

PCR run on samples from adult mosquitoes reared in cages contaminated with densonucleosis virus using primers specific for the virion proteins with an expected size of 372 bp.  Samples were taken up to 3 weeks post emergence. Lane 1 is the negative control.  Lanes 2 to 4 are the negative control cage samples. Lanes 5 to 10 and 13 to 18 are samples from infected cages. Lane 11 is the positive control.  Lane 12 is the 100 bp ladder with 100 bp at the bottom; the heavy band at the top is 500 bp.  Note that 6 of the 12 samples, those in lanes 6, 7, 10, 13, 14, and 16, are virion protein positive. (Erica Suchman, Colorado State University, Ft. Collins, CO)

PCR run on samples from adult mosquitoes reared in cages contaminated with densonucleosis virus using primers specific for the virion proteins with an expected size of 372 bp.  Samples were taken up to 3 weeks postemergence. Lane 1 is the negative control.  Lanes 2 to 4 are the negative control cage samples. Lanes 5 to 10 and 13 to 18 are samples from infected cages. Lane 11 is the positive control.  Lane 12 is the 100 bp ladder with 100 bp at the bottom; the heavy band at the top is 500 bp.  Note that 6 of the 12 samples, those in lanes 6, 7, 10, 13, 14, and 16, are virion protein positive. (Erica Suchman, Colorado State University, Ft. Collins, CO)

Variable number tandem repeat analysis of Mycobacterium bovis. Lanes 1 and 12 contain a 100 bp size ladder; the heavy band is 500 bp in length.  Lanes 2 to 9 contain isolates of M. bovis from different infected animals.  Lane 10 is the positive control that contains Mycobacterium tuberculosis H37Rv reference isolate. Lane 11 is the negative control. (Lorene Martinez, Colorado State University, Ft. Collins, CO)

Variable number tandem repeat analysis of Mycobacterium bovis. Lanes 1 and 12 contain a 100 bp size ladder.  Lanes 2 to 9 contain isolates of M. bovis from different infected animals.  Lane 10 is the positive control that contains Mycobacterium tuberculosis H37Rv reference isolate.  Lane 11 is the negative control.  Two genotypes are exhibited in this group of nine isolates. (Lorene Martinez, Colorado State University, Ft. Collins, CO)

Variable number tandem repeat analysis of Mycobacterium bovis. Lanes 1 and 12 contain a 100 bp size ladder.  Lanes 2 to 9 contain isolates of M. bovis from different infected animals.  Lane 10 is the positive control that contains Mycobacterium tuberculosis H37Rv reference isolate.  Lane 11 is the negative control.  One genotype is exhibited in this group of nine isolates. (Lorene Martinez, Colorado State University, Ft. Collins, CO)

Variable number tandem repeat analysis of Mycobacterium bovis. Lanes 1, 12, and 23 contain a 100 bp size ladder.  This gel represents nine isolates at two different loci. Locus 1: lanes 2 to 9 contain isolates of M. bovis from different infected animals.  Lane 10 is the positive control that contains Mycobacterium tuberculosis H37Rv reference isolate.  Lane 11 is the negative control.  Locus 2:  lanes 13 to 20 contain isolates of M. bovis from different infected animals.  Lane 21 is the positive control that contains Mycobacterium tuberculosis H37Rv reference isolate.  Lane 22 is the negative control.  One genotype is exhibited in this group of nine isolates at both loci. (Lorene Martinez, Colorado State University, Ft. Collins, CO)

Agarose gel electrophoresis of PCR products following real time Sybr green amplification.  Results for 12 different genes are shown. The far right lane is the 100 bp ladder, with 100 bp at the bottom; the heavy band is 500 bp.  Note the very faint band in lane 3 at ~300 bp, which is likely an amplification of genomic DNA as the intron separating the two exons for which the primers are specific is quite small.  gDNA digestion prior to reverse transcription can help to prevent this.  Additional small faint bands below primary amplicons are likely primer dimer or cDNA related and may be dismissed provided the dissociation curve displays only a single peak for each product.  Two percent agarose gel was stained with ethidium bromide and run for 90 minutes at 100 volts in 0.5x tris-borate-EDTA buffer, then visualized under UV light. (Liza O’Donoghue, Colorado State University, Ft. Collins, CO)

Standard multiplex PCR to assay species identity of cell lines.  Lanes 1 to 12 contain mouse cell lines from different contributors.  Lane 14 is the species specific ladder for six species of interest.  Lane 15 is the 100 bp ladder.  Lane 13 contains loading spillover from lane 14.  Two percent agarose gel was stained with ethidium bromide and run for 90 minutes at 100 volts in 1x tris-borate-EDTA buffer, then visualized under UV light. The far right lane is the 100 bp ladder, with 100 bp at the bottom; the heavy band is 500 bp. (Liza O’Donoghue, Colorado State University, Ft. Collins, CO)

The sequence of events in the PCR is as follows.  The temperature is raised to 92-98^oC, causing the DNA strands to separate.  Two primer sequences of approximately 20 nucleotides each are annealed to opposite strands of DNA.  (RNA requires an initial reverse transcription step to create a double-stranded cDNA template.)  The temperature is raised to the optimum for a polymerase from a thermophylic bacterium; usually Thermus aquaticus (Taq) is used at 72^oC.  Replication continues from the 3' OH of the primers, producing two copies of the DNA.  The temperature is again raised to 92-98^oC, causing the DNA strands to separate.  Then the temperature is lowered to allow new primers to attach to each of the four strands created in the previous reaction.  The temperature used during the annealing of primers must be optimized for each individual primer set.  The Taq polymerase fortunately is stable during the DNA melting step and is able to begin a

Whole blood from four shelter dogs was tested for canine distemper virus using real-time PCR. Case 1 sample and 1:10 dilution sample, pink and dark green lines, respectively; positive amplification control, light green line.  Cases 2 to 4, the negative extraction sample, and the amplification controls, 0 values.  A sample is considered positive when it crosses a fluorescence threshold of 30.  (Jeanette V. Bishop, Colorado State University, Ft. Collins, CO)

Taqman quantitative reverse transcription PCR analyzing mRNA levels of IFN-α5, IFN-β1, and IFN-γ in mouse spleens following challenge with aerosolized La Crosse virus.  Mice were either left untreated or received cationic liposome-DNA complex (CLDC) immunotherapy prior to virus challenge.  Both IFN-α5 and IFN-β1 mRNA production were significantly increased in the spleens of CLDC-treated mice at both days 2 and 4 postchallenge.  There were no significant differences between the production of IFN-γ mRNA in the spleens of untreated or treated mice.  The orange line is the chosen fluorescence threshold (cycle threshold or Ct value).  (Erik Arthun, Colorado State University, Ft. Collins, CO)

Quantitative reverse transcription PCR analysis showing the fold changes in IFN-α5 and IFN-β1 mRNA production between the spleens of untreated mice and mice treated with cationic liposome-DNA complex (CLDC) immunotherapy at days 2 and 4 postchallenge with aerosolized La Crosse virus.  Fold changes are relative to the levels of HPRT (a housekeeping control gene) in the spleens of untreated and unchallenged control mice.  Both IFN-α5 and IFN-β1 mRNA production were significantly increased in the spleens of CLDC-treated mice at both days 2 and 4 postchallenge. (Erik Arthun, Colorado State University, Ft. Collins, CO)

Taqman quantitative reverse transcription PCR analyzing mRNA levels of IL-12 and TNF-α in mouse spleens following challenge with aerosolized La Crosse virus.  Mice were either left untreated or received cationic liposome-DNA complex (CLDC) immunotherapy prior to virus challenge.  There were no significant differences between the production of IL-12 or TNF-α mRNA in the spleens of untreated or treated mice. The orange line is the chosen fluorescence threshold (cycle threshold or Ct value). (Erik Arthun, Colorado State University, Ft. Collins, CO)

Taqman quantitative reverse transcription PCR analyzing mRNA levels of IFN-γ, IL-12, and TNF-α in mouse brains following challenge with aerosolized La Crosse virus.  Mice were either left untreated or received cationic liposome-DNA complex (CLDC) immunotherapy prior to virus challenge.  IFN-γ mRNA production was significantly increased in the brains of CLDC-treated mice at both days 2 and 4 postchallenge.  There were no significant differences between the production of IL-12 and TNF-α mRNA in the brains of untreated or treated mice. The orange line is the chosen fluorescence threshold (cycle threshold or Ct value).  (Erik Arthun, Colorado State University, Ft. Collins, CO)

Quantitative reverse transcription PCR analysis showing the fold changes in IFN-γ mRNA production between the brains of untreated mice and mice treated with cationic liposome-DNA complex (CLDC) immunotherapy at days 2 and 4 postchallenge with aerosolized La Crosse virus. Fold changes are relative to the levels of HPRT (a housekeeping control gene) in the brains of untreated and unchallenged control mice.  IFN-γ mRNA production was significantly increased in the brains of CLDC-treated mice at both days 2 and 4 postchallenge.   (Erik Arthun, Colorado State University, Ft. Collins, CO)

Reverse transcription real-time Sybr green PCR of primary canine osteosarcoma tumors. Standard curve amplification plots for Sybr green analysis of HPRT1 expression on the Mx3000p instrument. This experiment was performed in duplicate using input cDNA quantities ranging from 100 ng to 5 ng equivalent RNA. Highest concentration samples amplify earliest (gold) with the lowest amplifying latest (blue).  Rox dye was used as a normalizer for PCR threshold (bottom flat lines). (Liza O’Donoghue, Colorado State University, Ft. Collins, CO)