METHOD FOR NORMALIZATION OF QUANTITATIVE PCR AND MICROARRAYS
The present invention concerns a method for comparing, in at least two samples A 1 and A 2 , the amount of RNA of a target gene t, wherein a fixed amount of external control sample C is added during RNA extraction of samples A 1 and A 2 and a fixed amount of external control D RNA is added to the extracted RNA from A 1 +C and A 2 +C before reverse transcription.
1 . A method for comparing, in at least two samples A 1 and A 2 , the amount of RNA of a target gene t, comprising the steps consisting of:
a) mixing each of the at least two samples A 1 and A 2 with a determined amount of external control sample C comprising RNA of a reference gene g c ;
b) extracting RNA from each of the at least two mixtures A 1 +C and A 2 +C obtained in step a), in order to obtain corresponding solutions of extracted RNA;
c) mixing each of the at least two solutions of extracted RNA of A 1 +C and A 2 +C with a determined amount of external control D RNA including RNA of a reference gene g d ;
d) performing reverse transcriptions on each of the at least two mixtures A 1 +C+D and A 2 +C+D obtained in step c), in order to obtain corresponding solutions comprising cDNAs of the target gene t, of the reference gene g c and of the reference gene g d ;
e) measuring the cDNA levels of each of the target gene t, of the reference gene g c and of the reference gene g d in each of the at least two cDNA solutions A 1 +C+D and A 2 +C+D obtained in step d); and
f) normalizing the cDNA levels of the target gene t from the at least two samples A 1 and A 2 , using cDNA levels of the reference genes g c and g d ;
wherein the reference gene g c is selected in such a way that nucleic acids, primers and/or probes used in step e) to measure the cDNA level of the reference gene g c do not cross-react with cDNAs of the target gene t and of the reference gene g d and wherein the reference gene g d is selected in such a way that nucleic acids, primers and/or probes used in step e) to measure the cDNA level of the reference gene g d do not cross-react with cDNAs of the target gene t and of the reference gene g c .
2 . The method according to claim 1 , wherein the reference genes g c and g d are selected in such a way that the order of magnitude of their relative expression level is similar to the order of magnitude of the relative expression level of the target gene t.
3 . The method according to claim 1 , wherein step e) of measuring cDNA levels is performed by quantitative PCR.
4 . The method according to claim 3 , wherein a cycle threshold Ct value is obtained in step e) for target gene t, and for reference genes g c and g d in each of the at least two cDNA solutions A 1 +C+D and A 2 +C+D.
5 . The method according to claim 4 , wherein step f) of normalizing is performed using the following equation:
R
=
2
-
[
Ct
(
t
A
1
)
-
Ct
(
g
c
A
1
)
]
+
Ct
(
g
d
A
2
)
-
Ct
(
g
d
A
1
)
2
-
[
Ct
(
t
A
2
)
-
Ct
(
g
c
A
2
)
]
;
wherein:
R represents the ratio of the cDNA level of the target gene t in the sample A 1 on the cDNA level of the target gene t in the sample A 2 ;
Ct(t A 1 ) represents the cycle threshold obtained for the target gene t in the mixture A 1 +C+D;
Ct(t A 2 ) represents the cycle threshold obtained for the target gene t in the mixture A 2 +C+D;
Ct(g c A 1 ) represents the cycle threshold obtained for the reference gene g c in the mixture A 1 +C+D;
Ct(g c A 2 ) represents the cycle threshold obtained for the reference gene g c in mixture A 2 +C+D;
Ct(g d A 1 ) represents the cycle threshold obtained for the reference gene g d in the mixture A 1 +C+D; and
Ct(g d A 2 ) represents the cycle threshold obtained for the reference gene g d in mixture A 2 +C+D.
6 . The method according to claim 1 , wherein the step e) of measuring cDNA levels is performed using microarrays.
7 . The method according to claim 6 , wherein a relative intensity fluorescence is obtained in step a) for target gene t, and for reference genes g c and g d in each of the at least two cDNA solutions A 1 +C+D and A 2 +C+D.
8 . A kit for comparing the amount of RNA of a target gene t in at least two samples A 1 and A 2 , comprising:
(i) a determined amount of an external control sample C comprising RNA of a reference gene g c and of a reference gene g′ c ;
(ii) a determined amount of external control D RNA including RNA of a reference gene g d and of a reference gene g′ d ;
(iii) a couple of primers that specifically amplify cDNA of the reference gene g c ;
(iv) a couple of primers that specifically amplify cDNA of the reference gene g′ c ;
(v) a couple of primers that specifically amplify cDNA of the reference gene g d ; and
(vi) a couple of primers that specifically amplify cDNA of the reference gene g′ d ;
wherein the reference genes g c and g d are genes with a relative low expression level and the reference genes g′ c and g′ d are genes with a relative high expression level.
9 . The kit according to claim 8 , wherein the couple of primers (iii), (iv), (v) and (vi) do not amplify cDNA of the target gene t.
10 . The method according to claim 2 , wherein step e) of measuring cDNA levels is performed by quantitative PCR.
11 . The method according to claim 2 , wherein the step e) of measuring cDNA levels is performed using microarrays.