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Bilateral Agreements
Reviewers' Checklists
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The Canadian Food Inspection Agency, Health Canada, and the United States
Department of Agriculture's Animal and Plant Health Inspection Service,
have prepared a series of checklists to be used by reviewers in the assessment
process for the following six analytical techniques: Southern blot, Western
blot, Northern blot, polymerase chain reaction, RNA dot blot, enzyme- linked
immunosorbent assay (ELISA), and enzyme assays. The agencies are sharing these
reviewers' checklists with potential applicants to provide guidance on the
preparation of quality data.
Checklist for Northern Blot Data |
Yes |
No |
Does
the Northern blot have a figure number and title? |
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Are
lanes labeled on the blot? |
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Does
the figure legend describe each lane of the blot, including a description of
the following for the RNA that was loaded:
- What type of material was loaded (eg. total purified
RNA, poly-A RNA, crude prep, total plant extract)?
- Source of the material loaded (eg. transformation event,
tissue, developmental stage, any prior treatments to induce gene expression,
etc.)
- Quantity of material loaded in each lane?
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Does
the text or figure legend describe how RNA was extracted prior to
electrophoresis? |
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Does
the blot have appropriate positive and negative control lanes (positive control
might demonstrate hybridization of the probe with itself; negative control
might be the unmodified parental plant line or variety)? |
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Is the
gel system and Northern hybridization protocol described in the text or in a
cited literature reference? Are any modifications of the cited protocols
described in the petition text? |
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Are the
position of molecular size standards on the gel indicated, and do they cover an
appropriate size range for the fragments that are expected to be detected on
the blot? |
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Is
there a description of the probe that was used for the hybridization? If so, is
the description adequate (in the text or in a figure) to enable one to
interpret the results? |
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If
quantitative analysis is performed, has the methodology or citation to such
been provided, and have a sufficient number of replicates or samples been
tested to determine whether there are significant differences between samples
or treatments? |
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Are any
superfluous bands or background signals properly explained? |
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Checklist for Southern Blot Data |
Yes |
No |
Does
the Southern blot have a figure number and title? |
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Are
lanes labeled on the blot? |
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Does
the figure legend describe each lane of the blot, including a description of
the following for the DNA that was loaded on the gel:
- Type of DNA loaded (eg. entire plasmid, restriction
fragment)?
- Source of DNA loaded (eg. transformation event,
tissue)?
- Restriction digestions of DNA prior to loading
gel?
- Quantity of material loaded in each lane?
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Does
the gel have appropriate positive and negative control lanes (positive control
might demonstrate hybridization of the probe with itself; negative control
might be the unmodified parental plant line or variety)? |
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Is the
gel system and Southern hybridization protocol described in the text or in a
cited literature reference? Are any modifications of the cited protocols
described in the petition text? |
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Are the
position of molecular size standards indicated, and do they cover an
appropriate size range for the fragments that are expected to be detected on
the blot? |
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Was an
entire plasmid used as the probe for the hybridization? If so, is the plasmid
described adequately in the text or in a figure to enable one to interpret the
results? |
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Was a
restriction fragment used as the probe for the hybridization? If so, is the
restriction fragment described adequately in the text or in a figure to enable
one to interpret the results? |
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Are any
superfluous bands or background signals properly explained? |
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Checklist for RNA Dot Blot Data |
Yes |
No |
Does
the dot blot have a figure number and title? |
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Are
lanes labeled on the blot? |
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Does
the figure legend describe each lane of the blot, including a description of
the following for the RNA that was loaded:
- What type of material was loaded (eg. total purified
RNA, poly-A RNA, crude prep, total plant extract)
- Source of the material loaded (e.g., transformation
event, tissue, developmental stage, any prior treatments to induce gene
expression, etc.)
- Quantity of material loaded in each lane?
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Does
the text or figure legend describe how RNA was extracted prior to blotting onto
the solid support? |
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Does
the blot have appropriate positive and negative control lanes (positive control
might demonstrate hybridization of the probe with itself; negative control
might be the unmodified parental plant line or variety)? |
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Are the
dot blot system and hybridization protocols described in the text or in a cited
literature reference? Are any modifications of the cited protocols described in
the submitted text? |
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Is
there a description of the probe that was used for the hybridization? If so, is
the description adequate (in the text or in a figure) to enable one to
interpret the results? |
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If
quantitative analysis is performed, has the methodology or citation to such
been provided, and have a sufficient number of replicates or samples been
tested to determine whether there are significant differences between samples
or treatments? |
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Checklist for Western Blot Data |
Yes |
No |
Does
the blot have a figure number and title? |
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Are the
lanes clearly labeled? |
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Does
the figure legend describe each lane of the blot, including a description of
the following for the protein that was loaded:
- Type of material loaded (eg. crude, pure, total
extract)?
- Source of material loaded (eg. Transformation event,
tissue type)?
- Quantity of material loaded?
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Is the
protein extraction method adequately described in either the text or the
figure? |
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Is the
antibody or antiserum preparation protocol adequately described in the text,
including an adequate description of the antigen and its purity? Has the
specificity of antibody or antiserum been determined and described in the text
or in a cited literature reference? |
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Is the
gel system and blotting protocol adequately described in the text or in a cited
literature reference? |
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Are the
position of molecular weight standards indicated, and do they cover the
appropriate range for the proteins expected to be detected on the
blot? |
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Does
the blot include appropriate positive and negative controls? |
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Was a
normal serum control conducted? |
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Are any
superfluous bands or background signal properly explained? |
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If
quantitative analysis is performed, has the methodology or citation to such
been provided, and have a sufficient number of replicates or samples been
tested to determine whether there are significant differences between samples
or treatments? |
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Checklist for PCR Data |
Yes |
No |
Does
the PCR gel have a figure number and title? |
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Are
lanes labeled on the gel? |
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Does
the figure legend describe each lane of the gel, including a description of the
following for the DNA that was loaded:
- What type of material was loaded (eg. plasmid fragment,
amplified DNA)?
- Source of material used in each reaction loaded (eg.
transformation event, tissue)?
- Quantity of material loaded?
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Are the
position of molecular weight standards indicated, and do they cover an
appropriate size range for the fragments that are expected to be detected on
gel? |
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Does
the text or figure legend describe how PCR amplification was performed prior to
electrophoresis? |
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Is
there a description of the primers used for amplification in the text or in the
figure sufficient to enable one to interpret the results? |
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Does
the gel have appropriate positive and negative control lanes (positive control
might demonstrate specificity of the primers and the ability to amplify the
appropriate size band; negative controls might include amplification with DNA
from the unmodified parental plant line or variety, and amplification in
absence of DNA template)? |
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Was an
entire plasmid or a restriction fragment used as the positive control template
and is it adequately described in the text or in the figure legend for
interpretation of the PCR results? |
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Is the
gel system and PCR protocol described in the text or in a cited literature
reference? Are modifications of a cited protocol described in the text?
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Checklist for ELISA Data |
Yes |
No |
Does
the table have a number and a title? |
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Are all
entries clearly identified in the table and described in the text or table
legend? |
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Is the
sample preparation method described? |
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Is the
antibody or antiserum preparation protocol adequately described in the text,
including a description of the antigen and its purity? Has the specificity of
antibody or antiserum been demonstrated and described in the text or in a cited
literature reference? |
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Is the
ELISA protocol used described in the text or cited in the scientific
literature? Any modifications to a cited protocol must be described.
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Were
appropriate positive controls (e.g. purified protein) and negative controls
(e.g. normal or preimmune serum, non-transformed plant material) used?
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When
ELISA is being used to quantify protein expression in transformed plant
tissues:
- Was a method for the determination of protein
concentration in tissue samples presented in the text or in a cited literature
reference?
- Was a standard curve prepared and the limit of detection
indicated?
- Have a sufficient number of replicates or samples been
tested to determine whether there are significant differences between samples
or treatments? Was statistical analysis performed?
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Checklist for Enzyme Assays |
Yes |
No |
Does
the figure (or table) have a number and title? |
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For
graphical representations or tables, are the axis or columns labeled and the
units indicated? |
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Does
the scale of the figure accurately represent and allow interpretation of the
data? |
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Does
the legend or text describe:
- The substrate and amount used for the
reaction?
- The quantity and origin of the enzyme?
- The temperature and pH?
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Does
the text or legend describe the extraction and purification of the enzyme and
the degree of purification achieved? |
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If the
enzyme used in the assay has not been isolated from the transformed plant but
is derived from an expression system, has adequate data been presented to
demonstrate its substantial equivalence to the plant expressed enzyme?
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Have
the assay method and relevant information concerning the enzyme been provided
in the text or in a cited literature reference? |
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Are
appropriate controls included in the assay? |
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Has the
stability of the enzyme and possible presence of enzyme inhibitors in different
tissue extracts been taken into account in the design of the assay or the
interpretation of the data? |
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When
relevant to the safety assessment, have the kinetics of the enzyme been
calculated and where possible compared to published data? |
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When
quantitative analysis is performed, have a sufficient number of replicates or
samples been tested to determine whether there are significant differences
between samples or treatments? Was statistical analysis performed? |
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