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Figure 1: PCR Run on Samples from Adult Mosquitoes Reared in Cages Contaminated with Densonucleosis Virus Using Primers Specific for the Virion Proteins. 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)

Figure 2: PCR Run on Samples from Adult Mosquitoes Reared in Cages Contaminated with Densonucleosis Virus Using Primers Specific for the Virion Proteins (Labeled view). 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)

Figure 3: PCR Run on Samples from Adult Mosquitoes Reared in Cages Contaminated with Densonucleosis Virus Using Primers Specific for the Virion Proteins. 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)

Figure 4: PCR Run on Samples from Adult Mosquitoes Reared in Cages Contaminated with Densonucleosis Virus Using Primers Specific for the Virion Proteins. 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)

Figure 5: PCR Run on Samples from Adult Mosquitoes Reared in Cages Contaminated with Densonucleosis Virus Using Primers Specific for the Virion Proteins. 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)

Figure 6: PCR Run on Samples from Adult Mosquitoes Reared in Cages Contaminated with Densonucleosis Virus Using Primers Specific for the Virion Proteins. 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)

Figure 7: Variable Number Tandem Repeat Analysis of Mycobacterium bovis. 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)

Figure 8: Variable Number Tandem Repeat Analysis of Mycobacterium bovis. 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)

Figure 9: Variable Number Tandem Repeat Analysis of Mycobacterium bovis. 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)

Figure 10: Variable Number Tandem Repeat Analysis of Mycobacterium bovis. 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)

Figure 11: Agarose Gel Electrophoresis of PCR Products Following Real Time Sybr Green Amplification. 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)

Figure 12: Standard Multiplex PCR to Assay Species Identity of Cell Lines. 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)

Figure 13: Reaction in PCR. The sequence of events in the PCR is as follows. The temperature is raised to 92-98oC, 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 72oC. Replication continues from the 3' OH of the primers, producing two copies of the DNA. The temperature is again raised to 92-98oC, 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 new cycle of synthesis. The process is repeated for 20 to 40 cycles so that additional copies arise exponentially, i.e., in a chain reaction. After amplification, the PCR product, sometimes called an amplicon, is analyzed on an agarose gel and is abundant enough to be detected with an ethidium bromide stain. The amplicon is compared to known sized molecular markers for production of bands of the correct size. (Erica Suchman, Colorado State University, Ft. Collins, CO)

Figure 14: Shelter Canines Tested for Canine Distemper Virus Using Real-Time PCR. 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)

Figure 15: Taqman Quantitative Reverse Transcription PCR. 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)

Figure 16: Quantitative Reverse Transcription PCR Analysis. 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)

Figure 17: Taqman Quantitative Reverse Transcription PCR. 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)

Figure 18: Taqman Quantitative Reverse Transcription PCR. 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)

Figure 19: Quantitative Reverse Transcription PCR Analysis. 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)

Figure 20: Reverse Transcription Real-Time Sybr Green PCR of Primary Canine Osteosarcoma Tumors. 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)

Figure 21: Reverse Transcription Real-Time Sybr Green PCR of Primary Canine Osteosarcoma Tumors. Reverse transcription real-time Sybr green PCR of primary canine osteosarcoma tumors. Standard curve output for Sybr green analysis of HPRT1 expression on the Mx3000p instrument. Standard curve analysis for each primer set is necessary for comparison of relative expression as differences in amplification efficiency can skew the data. 100% ± 10% amplification efficiency for each primer set is desirable; primer optimization is suggested outside of this range. This experiment was performed in duplicate using input cDNA quantities ranging from 100 ng to 5 ng equivalent RNA. (Liza O'Donoghue, Colorado State University, Ft. Collins, CO)

Figure 22: Reverse Transcription Real-Time Sybr Green PCR of Primary Canine Osteosarcoma Tumors. Reverse transcription real-time Sybr green PCR of primary canine osteosarcoma tumors. Dissociation curve for an ADHFE1 amplicon on the Mx3000p instrument (Sybr green). After the PCR cycles are completed, the samples are cooled to 55°C then ramped up to 94°C with the instrument collecting data continuously. The peak shows the temperature at which the PCR product melts.  If multiple products are present in the sample (e.g., due to nonspecific primers or gDNA contamination), multiple peaks will appear. Melting temperature is dependent upon amplicon size and G-C content, thus, only rarely do two different amplicons share the same melting temperature.  This test is not 100% accurate and all new primer sets should be validated by gel electrophoresis of the PCR products. (Liza O'Donoghue, Colorado State University, Ft. Collins, CO)

Figure 23: Reverse Transcription Real-Time Sybr Green PCR of Primary Canine Osteosarcoma Tumors. Reverse transcription real-time Sybr green PCR of primary canine osteosarcoma tumors. Dissociation curve for HPRT1 and a gene of interest (GOI). Note the two different primary peaks for the two different intended amplicons. Note also the aberrant peak at ~79°C.  This peak indicates the presence of an unintended product. As was the case here, peaks below 80°C are usually due to primer dimer formation secondary to insufficient template. This can be confirmed by gel electrophoresis. Nonetheless, any Sybr amplification for this sample must be considered faulty. (Liza O'Donoghue, Colorado State University, Ft. Collins, CO)

Figure 24: Real-Time Quantitative PCR. If quantification is desired one usually performs real-time quantitative PCR which requires the addition of a fluorescently labeled probe that hybridizes between the two primers (Taqman reverse transcription PCR) or the use of double-stranded DNA binding fluorescent dyes such as Syber green.  PCR amplification is usually quantified using a fluorescence detector, and the number of cycles of amplification required to cross a threshold fluorescence value (cycle threshold or CT) is determined by the computer and manipulated by the user.  The fewer the number of cycles required to cross the threshold, the more nucleic acids that are present in the sample.  CT values of unknown samples can be compared to CT values of known concentration standards to quantify the amount of nucleic acids in samples.  However, if one wants to quantify RNA, the RNA must first be reverse transcribed to cDNA and then used to perform real-time PCR.  The probe is complementary to sequences between the two primers used to amplify the DNA and contains a 5’ fluorescent reporter dye and a 3' quencher dye that disrupts (or quenches) the detectable signal from the fluorescent reporter dye when it is in close proximity via fluorescence resonance energy transfer.  As Taq polymerase polymerizes the DNA, its 5’ exonuclease activity will cleave the fluorescent dye from the primer liberating it.  As it floats away from the quencher dye its fluorescence will be detected by the detector. (Erica Suchman, Colorado State University, Ft. Collins, CO)

Figure 25: mecA PCR Product. Ethidium bromide-stained agarose mini-gel visualizing the products of multiple PCR reactions amplifying a portion of the mecA gene that encodes for methicillin resistance in Staphylococcus aureus ("MRSA"). Distinct 533 base-pair bands are clearly visible in the samples containing the resistance gene. Less distinct bands, indicating smaller primer dimers and unincorporated primers, are also visible. (Rebecca Buxton, University of Utah, Salt Lake City, UT)

Figure 26: mecA PCR Product (Labeled view). Ethidium bromide-stained agarose mini-gel visualizing the products of multiple PCR reactions amplifying a portion of the mecA gene that encodes for methicillin resistance in Staphylococcus aureus ("MRSA"). Distinct 533 base-pair bands are clearly visible in the samples containing the resistance gene. Less distinct bands, indicating smaller primer dimers and unincorporated primers, are also visible. (Labeled view) (Rebecca Buxton, University of Utah, Salt Lake City, UT)

Figure 27: Typical Temperature Program for a PCR Reaction. Typical temperature program for a PCR reaction. (Rebecca Buxton, University of Utah, Salt Lake City, UT)