Comparison between relative and absolute quantitative real-time PCR applied to single-cell analyses: Transcriptional levels in a key neuron for long-term memory in the pond snail.
Quantitative real-time PCR (qPCR) is a powerful method for measuring nucleic acid levels and quantifying mRNA levels, even in single cells. In the present study, we compared the results of single-cell qPCR obtained by different quantification methods (relative and absolute) and different reverse tra...
| Main Authors: | , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2022-01-01
|
| Series: | PLoS ONE |
| Online Access: | https://doi.org/10.1371/journal.pone.0279017 |
| _version_ | 1811177193973219328 |
|---|---|
| author | Dai Hatakeyama Nozomi Chikamoto Kanta Fujimoto Takashi Kitahashi Etsuro Ito |
| author_facet | Dai Hatakeyama Nozomi Chikamoto Kanta Fujimoto Takashi Kitahashi Etsuro Ito |
| author_sort | Dai Hatakeyama |
| collection | DOAJ |
| description | Quantitative real-time PCR (qPCR) is a powerful method for measuring nucleic acid levels and quantifying mRNA levels, even in single cells. In the present study, we compared the results of single-cell qPCR obtained by different quantification methods (relative and absolute) and different reverse transcription methods. In the experiments, we focused on the cerebral giant cell (CGC), a key neuron required for the acquisition of conditioned taste aversion in the pond snail Lymnaea stagnalis, and examined changes in the mRNA levels of 3 memory-related genes, cAMP-response element binding proteins (LymCREB1 and LymCREB2) and CREB-binding protein (LymCBP), during memory formation. The results obtained by relative quantification showed similar patterns for the 3 genes. For absolute quantification, reverse transcription was performed using 2 different methods: a mixture of oligo d(T) primers and random primers (RT method 1); and gene-specific primers (RT method 2). These methods yielded different results and did not show consistent changes related to conditioning. The mRNA levels in the samples prepared by RT method 2 were up to 3.3 times higher than those in samples prepared by RT method 1. These results suggest that for qPCR of single neurons, the efficacy and validity do not differ between relative and absolute quantification methods, but the reverse transcription step critically influences the results of mRNA quantification. |
| first_indexed | 2024-04-10T22:57:36Z |
| format | Article |
| id | doaj.art-106cfc62c6c5459ca3056bbaedf0e90e |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-04-10T22:57:36Z |
| publishDate | 2022-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-106cfc62c6c5459ca3056bbaedf0e90e2023-01-14T05:31:34ZengPublic Library of Science (PLoS)PLoS ONE1932-62032022-01-011712e027901710.1371/journal.pone.0279017Comparison between relative and absolute quantitative real-time PCR applied to single-cell analyses: Transcriptional levels in a key neuron for long-term memory in the pond snail.Dai HatakeyamaNozomi ChikamotoKanta FujimotoTakashi KitahashiEtsuro ItoQuantitative real-time PCR (qPCR) is a powerful method for measuring nucleic acid levels and quantifying mRNA levels, even in single cells. In the present study, we compared the results of single-cell qPCR obtained by different quantification methods (relative and absolute) and different reverse transcription methods. In the experiments, we focused on the cerebral giant cell (CGC), a key neuron required for the acquisition of conditioned taste aversion in the pond snail Lymnaea stagnalis, and examined changes in the mRNA levels of 3 memory-related genes, cAMP-response element binding proteins (LymCREB1 and LymCREB2) and CREB-binding protein (LymCBP), during memory formation. The results obtained by relative quantification showed similar patterns for the 3 genes. For absolute quantification, reverse transcription was performed using 2 different methods: a mixture of oligo d(T) primers and random primers (RT method 1); and gene-specific primers (RT method 2). These methods yielded different results and did not show consistent changes related to conditioning. The mRNA levels in the samples prepared by RT method 2 were up to 3.3 times higher than those in samples prepared by RT method 1. These results suggest that for qPCR of single neurons, the efficacy and validity do not differ between relative and absolute quantification methods, but the reverse transcription step critically influences the results of mRNA quantification.https://doi.org/10.1371/journal.pone.0279017 |
| spellingShingle | Dai Hatakeyama Nozomi Chikamoto Kanta Fujimoto Takashi Kitahashi Etsuro Ito Comparison between relative and absolute quantitative real-time PCR applied to single-cell analyses: Transcriptional levels in a key neuron for long-term memory in the pond snail. PLoS ONE |
| title | Comparison between relative and absolute quantitative real-time PCR applied to single-cell analyses: Transcriptional levels in a key neuron for long-term memory in the pond snail. |
| title_full | Comparison between relative and absolute quantitative real-time PCR applied to single-cell analyses: Transcriptional levels in a key neuron for long-term memory in the pond snail. |
| title_fullStr | Comparison between relative and absolute quantitative real-time PCR applied to single-cell analyses: Transcriptional levels in a key neuron for long-term memory in the pond snail. |
| title_full_unstemmed | Comparison between relative and absolute quantitative real-time PCR applied to single-cell analyses: Transcriptional levels in a key neuron for long-term memory in the pond snail. |
| title_short | Comparison between relative and absolute quantitative real-time PCR applied to single-cell analyses: Transcriptional levels in a key neuron for long-term memory in the pond snail. |
| title_sort | comparison between relative and absolute quantitative real time pcr applied to single cell analyses transcriptional levels in a key neuron for long term memory in the pond snail |
| url | https://doi.org/10.1371/journal.pone.0279017 |
| work_keys_str_mv | AT daihatakeyama comparisonbetweenrelativeandabsolutequantitativerealtimepcrappliedtosinglecellanalysestranscriptionallevelsinakeyneuronforlongtermmemoryinthepondsnail AT nozomichikamoto comparisonbetweenrelativeandabsolutequantitativerealtimepcrappliedtosinglecellanalysestranscriptionallevelsinakeyneuronforlongtermmemoryinthepondsnail AT kantafujimoto comparisonbetweenrelativeandabsolutequantitativerealtimepcrappliedtosinglecellanalysestranscriptionallevelsinakeyneuronforlongtermmemoryinthepondsnail AT takashikitahashi comparisonbetweenrelativeandabsolutequantitativerealtimepcrappliedtosinglecellanalysestranscriptionallevelsinakeyneuronforlongtermmemoryinthepondsnail AT etsuroito comparisonbetweenrelativeandabsolutequantitativerealtimepcrappliedtosinglecellanalysestranscriptionallevelsinakeyneuronforlongtermmemoryinthepondsnail |