Setting Up Reverse Transcription Quantitative-PCR Experiments View Full Text


Ontology type: schema:Chapter     


Chapter Info

DATE

2010-09-07

AUTHORS

Madana M. R. Ambavaram , Andy Pereira

ABSTRACT

Quantitative real-time PCR (qRT-PCR), in conjunction with reverse transcriptase, has been used for the systematic measurement of plant physiological changes in gene expression. In the present paper, we describe a qRT-PCR protocol that illustrates the essential technical steps required to generate quantitative data that are reliable and reproducible. To demonstrate the methods used, we evaluated the expression stability of five [actin (ACT), actin1 (ACT1), β-glyceraldehyde-3-phosphate dehydrogenase (GAPDH), cyclophilin (CYC), and elongation factor 1α (EF-1α)] frequently used housekeeping genes in rice. The expression stability of the five selected housekeeping genes varied considerably in different tissues (seedlings, vegetative and reproductive stages) in a given stress condition. The analysis allowed us to choose a set of two candidates (ACT1 and EF-1α) that showed more uniform expression and are also suitable for the validation of weakly expressed genes (≥0.5 fold), identified through microarray analysis. More... »

PAGES

45-54

Identifiers

URI

http://scigraph.springernature.com/pub.10.1007/978-1-60761-682-5_4

DOI

http://dx.doi.org/10.1007/978-1-60761-682-5_4

DIMENSIONS

https://app.dimensions.ai/details/publication/pub.1013411701

PUBMED

https://www.ncbi.nlm.nih.gov/pubmed/20931371


Indexing Status Check whether this publication has been indexed by Scopus and Web Of Science using the SN Indexing Status Tool
Incoming Citations Browse incoming citations for this publication using opencitations.net

JSON-LD is the canonical representation for SciGraph data.

TIP: You can open this SciGraph record using an external JSON-LD service: JSON-LD Playground Google SDTT

[
  {
    "@context": "https://springernature.github.io/scigraph/jsonld/sgcontext.json", 
    "about": [
      {
        "id": "http://purl.org/au-research/vocabulary/anzsrc-for/2008/06", 
        "inDefinedTermSet": "http://purl.org/au-research/vocabulary/anzsrc-for/2008/", 
        "name": "Biological Sciences", 
        "type": "DefinedTerm"
      }, 
      {
        "id": "http://purl.org/au-research/vocabulary/anzsrc-for/2008/0604", 
        "inDefinedTermSet": "http://purl.org/au-research/vocabulary/anzsrc-for/2008/", 
        "name": "Genetics", 
        "type": "DefinedTerm"
      }, 
      {
        "inDefinedTermSet": "https://www.nlm.nih.gov/mesh/", 
        "name": "Actins", 
        "type": "DefinedTerm"
      }, 
      {
        "inDefinedTermSet": "https://www.nlm.nih.gov/mesh/", 
        "name": "Cyclophilins", 
        "type": "DefinedTerm"
      }, 
      {
        "inDefinedTermSet": "https://www.nlm.nih.gov/mesh/", 
        "name": "Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating)", 
        "type": "DefinedTerm"
      }, 
      {
        "inDefinedTermSet": "https://www.nlm.nih.gov/mesh/", 
        "name": "Oryza", 
        "type": "DefinedTerm"
      }, 
      {
        "inDefinedTermSet": "https://www.nlm.nih.gov/mesh/", 
        "name": "Peptide Elongation Factor 1", 
        "type": "DefinedTerm"
      }, 
      {
        "inDefinedTermSet": "https://www.nlm.nih.gov/mesh/", 
        "name": "Plant Proteins", 
        "type": "DefinedTerm"
      }, 
      {
        "inDefinedTermSet": "https://www.nlm.nih.gov/mesh/", 
        "name": "Reverse Transcriptase Polymerase Chain Reaction", 
        "type": "DefinedTerm"
      }
    ], 
    "author": [
      {
        "affiliation": {
          "alternateName": "Virginia Bioinformatics Institute, Virginia Tech, Blacksburg, VA, USA", 
          "id": "http://www.grid.ac/institutes/grid.438526.e", 
          "name": [
            "Virginia Bioinformatics Institute, Virginia Tech, Blacksburg, VA, USA"
          ], 
          "type": "Organization"
        }, 
        "familyName": "Ambavaram", 
        "givenName": "Madana M. R.", 
        "id": "sg:person.01000002361.29", 
        "sameAs": [
          "https://app.dimensions.ai/discover/publication?and_facet_researcher=ur.01000002361.29"
        ], 
        "type": "Person"
      }, 
      {
        "affiliation": {
          "alternateName": "Virginia Bioinformatics Institute, Virginia Tech, Blacksburg, VA, USA", 
          "id": "http://www.grid.ac/institutes/grid.438526.e", 
          "name": [
            "Virginia Bioinformatics Institute, Virginia Tech, Blacksburg, VA, USA"
          ], 
          "type": "Organization"
        }, 
        "familyName": "Pereira", 
        "givenName": "Andy", 
        "id": "sg:person.01344430566.76", 
        "sameAs": [
          "https://app.dimensions.ai/discover/publication?and_facet_researcher=ur.01344430566.76"
        ], 
        "type": "Person"
      }
    ], 
    "datePublished": "2010-09-07", 
    "datePublishedReg": "2010-09-07", 
    "description": "Quantitative real-time PCR (qRT-PCR), in conjunction with reverse transcriptase, has been used for the systematic measurement of plant physiological changes in gene expression. In the present paper, we describe a qRT-PCR protocol that illustrates the essential technical steps required to generate quantitative data that are reliable and reproducible. To demonstrate the methods used, we evaluated the expression stability of five [actin (ACT), actin1 (ACT1), \u03b2-glyceraldehyde-3-phosphate dehydrogenase (GAPDH), cyclophilin (CYC), and elongation factor 1\u03b1 (EF-1\u03b1)] frequently used housekeeping genes in rice. The expression stability of the five selected housekeeping genes varied considerably in different tissues (seedlings, vegetative and reproductive stages) in a given stress condition. The analysis allowed us to choose a set of two candidates (ACT1 and EF-1\u03b1) that showed more uniform expression and are also suitable for the validation of weakly expressed genes (\u22650.5 fold), identified through microarray analysis.", 
    "editor": [
      {
        "familyName": "Pereira", 
        "givenName": "Andy", 
        "type": "Person"
      }
    ], 
    "genre": "chapter", 
    "id": "sg:pub.10.1007/978-1-60761-682-5_4", 
    "inLanguage": "en", 
    "isAccessibleForFree": false, 
    "isPartOf": {
      "isbn": [
        "978-1-60761-681-8", 
        "978-1-60761-682-5"
      ], 
      "name": "Plant Reverse Genetics", 
      "type": "Book"
    }, 
    "keywords": [
      "housekeeping genes", 
      "expression stability", 
      "quantitative real-time PCR", 
      "plant physiological changes", 
      "qRT-PCR protocol", 
      "real-time PCR", 
      "gene expression", 
      "microarray analysis", 
      "stress conditions", 
      "genes", 
      "different tissues", 
      "reverse transcriptase", 
      "uniform expression", 
      "physiological changes", 
      "expression", 
      "more uniform expression", 
      "rice", 
      "PCR", 
      "transcriptase", 
      "tissue", 
      "analysis", 
      "step", 
      "changes", 
      "candidates", 
      "quantitative data", 
      "stability", 
      "experiments", 
      "conditions", 
      "data", 
      "technical steps", 
      "set", 
      "conjunction", 
      "protocol", 
      "validation", 
      "present paper", 
      "method", 
      "systematic measurements", 
      "measurements", 
      "paper", 
      "essential technical steps", 
      "Reverse Transcription Quantitative-PCR Experiments", 
      "Transcription Quantitative-PCR Experiments", 
      "Quantitative-PCR Experiments"
    ], 
    "name": "Setting Up Reverse Transcription Quantitative-PCR Experiments", 
    "pagination": "45-54", 
    "productId": [
      {
        "name": "dimensions_id", 
        "type": "PropertyValue", 
        "value": [
          "pub.1013411701"
        ]
      }, 
      {
        "name": "doi", 
        "type": "PropertyValue", 
        "value": [
          "10.1007/978-1-60761-682-5_4"
        ]
      }, 
      {
        "name": "pubmed_id", 
        "type": "PropertyValue", 
        "value": [
          "20931371"
        ]
      }
    ], 
    "publisher": {
      "name": "Springer Nature", 
      "type": "Organisation"
    }, 
    "sameAs": [
      "https://doi.org/10.1007/978-1-60761-682-5_4", 
      "https://app.dimensions.ai/details/publication/pub.1013411701"
    ], 
    "sdDataset": "chapters", 
    "sdDatePublished": "2021-11-01T18:51", 
    "sdLicense": "https://scigraph.springernature.com/explorer/license/", 
    "sdPublisher": {
      "name": "Springer Nature - SN SciGraph project", 
      "type": "Organization"
    }, 
    "sdSource": "s3://com-springernature-scigraph/baseset/20211101/entities/gbq_results/chapter/chapter_229.jsonl", 
    "type": "Chapter", 
    "url": "https://doi.org/10.1007/978-1-60761-682-5_4"
  }
]
 

Download the RDF metadata as:  json-ld nt turtle xml License info

HOW TO GET THIS DATA PROGRAMMATICALLY:

JSON-LD is a popular format for linked data which is fully compatible with JSON.

curl -H 'Accept: application/ld+json' 'https://scigraph.springernature.com/pub.10.1007/978-1-60761-682-5_4'

N-Triples is a line-based linked data format ideal for batch operations.

curl -H 'Accept: application/n-triples' 'https://scigraph.springernature.com/pub.10.1007/978-1-60761-682-5_4'

Turtle is a human-readable linked data format.

curl -H 'Accept: text/turtle' 'https://scigraph.springernature.com/pub.10.1007/978-1-60761-682-5_4'

RDF/XML is a standard XML format for linked data.

curl -H 'Accept: application/rdf+xml' 'https://scigraph.springernature.com/pub.10.1007/978-1-60761-682-5_4'


 

This table displays all metadata directly associated to this object as RDF triples.

142 TRIPLES      23 PREDICATES      76 URIs      69 LITERALS      15 BLANK NODES

Subject Predicate Object
1 sg:pub.10.1007/978-1-60761-682-5_4 schema:about N0cc4cfab32b04399aeda7c434199b5a8
2 N4c6c110d65a64d20b6842c03e06b4eaa
3 N52a6cf09dee2481aa7ceb4c63f71a3d6
4 N839c8463e9c749cc9f0e2ec5c9522d24
5 Nd3db15409221429eadc027afb5bc8c60
6 Nddec450f19cc4d4181bfd9da77a2a3fe
7 Ne648730d7cfd4bf4b0a86d3e9a1d81de
8 anzsrc-for:06
9 anzsrc-for:0604
10 schema:author N5d85c486e165491bb7d0af13d7ca42ea
11 schema:datePublished 2010-09-07
12 schema:datePublishedReg 2010-09-07
13 schema:description Quantitative real-time PCR (qRT-PCR), in conjunction with reverse transcriptase, has been used for the systematic measurement of plant physiological changes in gene expression. In the present paper, we describe a qRT-PCR protocol that illustrates the essential technical steps required to generate quantitative data that are reliable and reproducible. To demonstrate the methods used, we evaluated the expression stability of five [actin (ACT), actin1 (ACT1), β-glyceraldehyde-3-phosphate dehydrogenase (GAPDH), cyclophilin (CYC), and elongation factor 1α (EF-1α)] frequently used housekeeping genes in rice. The expression stability of the five selected housekeeping genes varied considerably in different tissues (seedlings, vegetative and reproductive stages) in a given stress condition. The analysis allowed us to choose a set of two candidates (ACT1 and EF-1α) that showed more uniform expression and are also suitable for the validation of weakly expressed genes (≥0.5 fold), identified through microarray analysis.
14 schema:editor N06f23cd197044783bd94f51313c50e05
15 schema:genre chapter
16 schema:inLanguage en
17 schema:isAccessibleForFree false
18 schema:isPartOf Nf26b466c4d9d472ab319b851c2af83ed
19 schema:keywords PCR
20 Quantitative-PCR Experiments
21 Reverse Transcription Quantitative-PCR Experiments
22 Transcription Quantitative-PCR Experiments
23 analysis
24 candidates
25 changes
26 conditions
27 conjunction
28 data
29 different tissues
30 essential technical steps
31 experiments
32 expression
33 expression stability
34 gene expression
35 genes
36 housekeeping genes
37 measurements
38 method
39 microarray analysis
40 more uniform expression
41 paper
42 physiological changes
43 plant physiological changes
44 present paper
45 protocol
46 qRT-PCR protocol
47 quantitative data
48 quantitative real-time PCR
49 real-time PCR
50 reverse transcriptase
51 rice
52 set
53 stability
54 step
55 stress conditions
56 systematic measurements
57 technical steps
58 tissue
59 transcriptase
60 uniform expression
61 validation
62 schema:name Setting Up Reverse Transcription Quantitative-PCR Experiments
63 schema:pagination 45-54
64 schema:productId N1df179cc34b346ddbfca66a3920e88d7
65 N285979ecf96f43f8b60f455baa5ec709
66 Nb79586bc76244ef093b7ef8fb87817b5
67 schema:publisher N3bf2f851e3904fc68a5cc7e8b80712c0
68 schema:sameAs https://app.dimensions.ai/details/publication/pub.1013411701
69 https://doi.org/10.1007/978-1-60761-682-5_4
70 schema:sdDatePublished 2021-11-01T18:51
71 schema:sdLicense https://scigraph.springernature.com/explorer/license/
72 schema:sdPublisher N9ece07a04cdb48fe8297adbd1af9e909
73 schema:url https://doi.org/10.1007/978-1-60761-682-5_4
74 sgo:license sg:explorer/license/
75 sgo:sdDataset chapters
76 rdf:type schema:Chapter
77 N06f23cd197044783bd94f51313c50e05 rdf:first N6156bd603fc54cccbe7de73f60102e7c
78 rdf:rest rdf:nil
79 N0cc4cfab32b04399aeda7c434199b5a8 schema:inDefinedTermSet https://www.nlm.nih.gov/mesh/
80 schema:name Cyclophilins
81 rdf:type schema:DefinedTerm
82 N1df179cc34b346ddbfca66a3920e88d7 schema:name doi
83 schema:value 10.1007/978-1-60761-682-5_4
84 rdf:type schema:PropertyValue
85 N285979ecf96f43f8b60f455baa5ec709 schema:name dimensions_id
86 schema:value pub.1013411701
87 rdf:type schema:PropertyValue
88 N3bf2f851e3904fc68a5cc7e8b80712c0 schema:name Springer Nature
89 rdf:type schema:Organisation
90 N4c6c110d65a64d20b6842c03e06b4eaa schema:inDefinedTermSet https://www.nlm.nih.gov/mesh/
91 schema:name Oryza
92 rdf:type schema:DefinedTerm
93 N52a6cf09dee2481aa7ceb4c63f71a3d6 schema:inDefinedTermSet https://www.nlm.nih.gov/mesh/
94 schema:name Reverse Transcriptase Polymerase Chain Reaction
95 rdf:type schema:DefinedTerm
96 N5d85c486e165491bb7d0af13d7ca42ea rdf:first sg:person.01000002361.29
97 rdf:rest Nfdaf6e08ee074fb9af2d60a945e71a6f
98 N6156bd603fc54cccbe7de73f60102e7c schema:familyName Pereira
99 schema:givenName Andy
100 rdf:type schema:Person
101 N839c8463e9c749cc9f0e2ec5c9522d24 schema:inDefinedTermSet https://www.nlm.nih.gov/mesh/
102 schema:name Actins
103 rdf:type schema:DefinedTerm
104 N9ece07a04cdb48fe8297adbd1af9e909 schema:name Springer Nature - SN SciGraph project
105 rdf:type schema:Organization
106 Nb79586bc76244ef093b7ef8fb87817b5 schema:name pubmed_id
107 schema:value 20931371
108 rdf:type schema:PropertyValue
109 Nd3db15409221429eadc027afb5bc8c60 schema:inDefinedTermSet https://www.nlm.nih.gov/mesh/
110 schema:name Plant Proteins
111 rdf:type schema:DefinedTerm
112 Nddec450f19cc4d4181bfd9da77a2a3fe schema:inDefinedTermSet https://www.nlm.nih.gov/mesh/
113 schema:name Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating)
114 rdf:type schema:DefinedTerm
115 Ne648730d7cfd4bf4b0a86d3e9a1d81de schema:inDefinedTermSet https://www.nlm.nih.gov/mesh/
116 schema:name Peptide Elongation Factor 1
117 rdf:type schema:DefinedTerm
118 Nf26b466c4d9d472ab319b851c2af83ed schema:isbn 978-1-60761-681-8
119 978-1-60761-682-5
120 schema:name Plant Reverse Genetics
121 rdf:type schema:Book
122 Nfdaf6e08ee074fb9af2d60a945e71a6f rdf:first sg:person.01344430566.76
123 rdf:rest rdf:nil
124 anzsrc-for:06 schema:inDefinedTermSet anzsrc-for:
125 schema:name Biological Sciences
126 rdf:type schema:DefinedTerm
127 anzsrc-for:0604 schema:inDefinedTermSet anzsrc-for:
128 schema:name Genetics
129 rdf:type schema:DefinedTerm
130 sg:person.01000002361.29 schema:affiliation grid-institutes:grid.438526.e
131 schema:familyName Ambavaram
132 schema:givenName Madana M. R.
133 schema:sameAs https://app.dimensions.ai/discover/publication?and_facet_researcher=ur.01000002361.29
134 rdf:type schema:Person
135 sg:person.01344430566.76 schema:affiliation grid-institutes:grid.438526.e
136 schema:familyName Pereira
137 schema:givenName Andy
138 schema:sameAs https://app.dimensions.ai/discover/publication?and_facet_researcher=ur.01344430566.76
139 rdf:type schema:Person
140 grid-institutes:grid.438526.e schema:alternateName Virginia Bioinformatics Institute, Virginia Tech, Blacksburg, VA, USA
141 schema:name Virginia Bioinformatics Institute, Virginia Tech, Blacksburg, VA, USA
142 rdf:type schema:Organization
 




Preview window. Press ESC to close (or click here)


...