Limits...
Quantitative RT-PCR assay for high-throughput screening (HTS) of drugs against the growth of Cryptosporidium parvum in vitro.

Zhang H, Zhu G - Front Microbiol (2015)

Bottom Line: However, conventional qRT-PCR protocol is not very amendable to high-throughput analysis when total RNA needs to be purified by lengthy, multi-step procedures.Recently, several commercial reagents are available for preparing cell lysates that could be directly used in downstream qRT-PCR analysis (e.g., Ambion Cell-to-cDNA kit and Bio-Rad iScript sample preparation reagent).This qRT-PCR protocol is able to give a >150-fold linear dynamic range using samples isolated from cells infected with various numbers of parasites.

View Article: PubMed Central - PubMed

Affiliation: Department of Veterinary Pathobiology, College of Veterinary Medicine & Biomedical Sciences, Texas A&M University College Station, TX, USA.

ABSTRACT
Our laboratory has previously developed a qRT-PCR assay to assess drug efficacy on the growth of Cryptosporidium parvum in vitro by detecting the levels of parasite 18S rRNA. This approach displayed up to four orders of magnitude of linear dynamic range and was much less labor-intensive than the traditional microscopic methods. However, conventional qRT-PCR protocol is not very amendable to high-throughput analysis when total RNA needs to be purified by lengthy, multi-step procedures. Recently, several commercial reagents are available for preparing cell lysates that could be directly used in downstream qRT-PCR analysis (e.g., Ambion Cell-to-cDNA kit and Bio-Rad iScript sample preparation reagent). Using these reagents, we are able to adapt the qRT-PCR assay into high-throughput screening of drugs in vitro (i.e., 96-well and 384-well formats for the cultivation of parasites and qRT-PCR detection, respectively). This qRT-PCR protocol is able to give a >150-fold linear dynamic range using samples isolated from cells infected with various numbers of parasites. The new assay is also validated by the NIH-recommended intra-plate, inter-plate, and inter-day uniformity tests. The robustness and effectiveness of the assay are also confirmed by evaluating the anti-cryptosporidial efficacy of paromomycin and by a small scale screening of compounds.

No MeSH data available.


Specific detection of Cp18S and Hs18S rRNA transcripts as demonstrated by amplification curve (A) and melting curve (B) analyses of a representative plate. Lysates of HCT-8 cells grown in 96-well plates and infected with Cryptosporidium parvum for 44 h were used in qRT-PCR reactions in 384-well format.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4585199&req=5

Figure 3: Specific detection of Cp18S and Hs18S rRNA transcripts as demonstrated by amplification curve (A) and melting curve (B) analyses of a representative plate. Lysates of HCT-8 cells grown in 96-well plates and infected with Cryptosporidium parvum for 44 h were used in qRT-PCR reactions in 384-well format.

Mentions: The subsequent qRT-PCR amplification protocol in 384-well format using cell lysates was effective and specific based on agarose gel analysis of amplicons (data not shown) and post-run analysis of amplification and melting curves (Figure 3). In the melting curves, Cp18S and Hs18S displayed two strong and distinguishable peaks, although there was a small minor peak in the Cp18S curves (Figure 3B).


Quantitative RT-PCR assay for high-throughput screening (HTS) of drugs against the growth of Cryptosporidium parvum in vitro.

Zhang H, Zhu G - Front Microbiol (2015)

Specific detection of Cp18S and Hs18S rRNA transcripts as demonstrated by amplification curve (A) and melting curve (B) analyses of a representative plate. Lysates of HCT-8 cells grown in 96-well plates and infected with Cryptosporidium parvum for 44 h were used in qRT-PCR reactions in 384-well format.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4585199&req=5

Figure 3: Specific detection of Cp18S and Hs18S rRNA transcripts as demonstrated by amplification curve (A) and melting curve (B) analyses of a representative plate. Lysates of HCT-8 cells grown in 96-well plates and infected with Cryptosporidium parvum for 44 h were used in qRT-PCR reactions in 384-well format.
Mentions: The subsequent qRT-PCR amplification protocol in 384-well format using cell lysates was effective and specific based on agarose gel analysis of amplicons (data not shown) and post-run analysis of amplification and melting curves (Figure 3). In the melting curves, Cp18S and Hs18S displayed two strong and distinguishable peaks, although there was a small minor peak in the Cp18S curves (Figure 3B).

Bottom Line: However, conventional qRT-PCR protocol is not very amendable to high-throughput analysis when total RNA needs to be purified by lengthy, multi-step procedures.Recently, several commercial reagents are available for preparing cell lysates that could be directly used in downstream qRT-PCR analysis (e.g., Ambion Cell-to-cDNA kit and Bio-Rad iScript sample preparation reagent).This qRT-PCR protocol is able to give a >150-fold linear dynamic range using samples isolated from cells infected with various numbers of parasites.

View Article: PubMed Central - PubMed

Affiliation: Department of Veterinary Pathobiology, College of Veterinary Medicine & Biomedical Sciences, Texas A&M University College Station, TX, USA.

ABSTRACT
Our laboratory has previously developed a qRT-PCR assay to assess drug efficacy on the growth of Cryptosporidium parvum in vitro by detecting the levels of parasite 18S rRNA. This approach displayed up to four orders of magnitude of linear dynamic range and was much less labor-intensive than the traditional microscopic methods. However, conventional qRT-PCR protocol is not very amendable to high-throughput analysis when total RNA needs to be purified by lengthy, multi-step procedures. Recently, several commercial reagents are available for preparing cell lysates that could be directly used in downstream qRT-PCR analysis (e.g., Ambion Cell-to-cDNA kit and Bio-Rad iScript sample preparation reagent). Using these reagents, we are able to adapt the qRT-PCR assay into high-throughput screening of drugs in vitro (i.e., 96-well and 384-well formats for the cultivation of parasites and qRT-PCR detection, respectively). This qRT-PCR protocol is able to give a >150-fold linear dynamic range using samples isolated from cells infected with various numbers of parasites. The new assay is also validated by the NIH-recommended intra-plate, inter-plate, and inter-day uniformity tests. The robustness and effectiveness of the assay are also confirmed by evaluating the anti-cryptosporidial efficacy of paromomycin and by a small scale screening of compounds.

No MeSH data available.