KR20120107717A - Method for selecting a threshold in amplification profile curve of real-time pcr - Google Patents

Abstract

PURPOSE: A method for selecting a threshold from an amplification profile curve of a real time polymerase chain reaction is provided to more accurately select a threshold value for the amount of polynucleotide which does not use a reference curve. CONSTITUTION: Polynucleotide polymerase chain reaction for a plurality of samples including two or more of the same samples is performed. An amplification profile curve for signal strength values which are provided by probes is obtained by every sample. One threshold value is selected among the signal strength values. A threshold cycle value corresponding to the selected threshold value is calculated. A deviation value of threshold cycle values is calculated. [Reference numerals] (AA) Stagnant state area; (BB) Exponential area; (CC) Probe signal strength; (DD) Base line area; (EE) Amplification cycle number; (FF) Amplification profile curve in general RT PCR

Description

Method for selecting a threshold in amplification profile curve of real-time PCR}

The present invention relates to a method for selecting a threshold for polynucleotide quantification from an amplification profile curve obtained by performing a real time polymerase chain reaction.

Real-time polynucleotide chain reaction (real-time PCR) is a technology that can quantify polynucleotides by monitoring the increase of PCR amplification products in real time. It is widely used in biological research and clinical analysis such as monitoring, genomic-level gene quantification and pathogen detection.

In real-time PCR, the amount of PCR amplification product can be detected by fluorescence signal. Intercalating method using a reagent that shows fluorescence by binding to double-stranded DNA as a detection method, a method of using oligonucleotides labeled with a fluorescent material at the 5 'end and a quencher material at the 3' end, etc. There is this. When the above methods are used, the intensity of the fluorescence signal increases according to the amount of polynucleotide that increases with real-time PCR, and the user generates an amplification profile curve indicating the intensity of the fluorescence signal according to the number of amplification cycles. You get

The amplification profile curve is typically a baseline region where the background fluorescence signal intensity does not reflect the actual amount of polynucleotides, an exponential region where the increase in fluorescence signal intensity is reflected by the increase in the amount of polynucleotides, And the PCR reaction is saturated and divided into a plateau region where no further increase in fluorescence signal intensity appears (FIG. 1). The baseline region appears early in the PCR reaction because the amount of PCR amplification products has not yet reached a detectable amount.

Typically, the fluorescence signal intensity at the point of transition from the baseline region to the exponential region, that is, when the amount of PCR amplification products reaches a detectable amount by fluorescence, is called a threshold and corresponds to a threshold in the amplification profile curve. The number of amplification cycles is called a threshold cycle (CT) value. When real-time PCR was performed at different initial polynucleotide concentrations, the larger the initial polynucleotide amount, the smaller the number of amplification cycles in which the amount of amplification products reached a detectable amount. Therefore, the log value of the initial polynucleotide amount and the threshold cycle value are in inversely strong relationship, and typically, the desired polynucleotide quantification is performed using the threshold cycle value. Polynucleotide quantification using real-time polymerase chain reaction has large absolute and relative quantification. Absolute quantification is a method of quantifying by generating a standard curve of critical cycle values for a polynucleotide amount by performing a real-time polymerase chain reaction with a sample having a known polynucleotide amount. Even if you do not know the amount, it is a way to compare the relative amount with other samples. For relative quantification, standard curves may or may not be used. In all these cases, in order to quantify, the threshold value must first be determined to calculate the threshold cycle value.

A method for determining the threshold value is a threshold value when the fluorescence signal intensity reaches a predetermined signal level called AFL (arbitrary fluorescence value), and the maximum value of the second derivative of the amplification profile curve And a threshold value. The method using AFL has the disadvantage of being sensitive to the change in the average baseline fluorescence level in the initial PCR cycle, and the method using the derivative has the disadvantage of being sensitive to noise and separately analyzing each of the amplification profile curves. If these methods do not produce a suitable standard curve or produce an unreliable quantitative result, the user can typically visually determine the threshold value, even in this case because the subjective judgment of the user is used to determine the quantification of the polynucleotide. Can lower the accuracy. Therefore, there is a need to select the correct threshold from the amplification profile curve data of real-time PCR for more accurate polynucleotide quantification.

The present invention provides a method for more accurate polynucleotide quantification.

The present invention provides a method for selecting a more accurate threshold in the amplification profile curve of a real time polymerase chain reaction.

 The present invention provides a method for selecting a more accurate threshold for polynucleotide quantification that does not use a standard curve.

A first aspect of the present invention is a method of selecting a threshold value from an amplification profile curve obtained by performing a real-time polymerase chain reaction.

(a) performing a polynucleotide polymerase chain reaction on a plurality of samples including two or more identical samples, wherein the polymerase chain reaction includes a plurality of amplification cycles and provides a signal according to the amount of polynucleotides. Performing in the presence of a detectable probe that can provide;

(b) obtaining an amplification profile curve for the signal strength value provided by the probe according to the number of amplification cycles for each sample;

(c) selecting one threshold from the signal strength values;

(d) calculating a threshold cycle value corresponding to the selected threshold value from the amplification profile curves;

(e) calculating a variance value of threshold cycle values of the same samples;

(f) repeating steps (c), (d), and (e) a plurality of times by varying the threshold value to be selected; And

(g) selecting the threshold value of step (c) of calculating the smallest variance value among the variance values calculated in step (e) as a final threshold value.

The method selects a threshold value from an amplification profile curve obtained by performing a real-time polymerase chain reaction.

The term "real-time polymerase chain reaction (RT PCR)" is an improvement on the polymerase chain reaction (PCR) that amplifies polynucleotides using polymerases. It refers to a technology that can monitor in real time the increase in the amount of polynucleotide amplified by adding a fluorescent material capable of binding to the polynucleotide by the intensity of the fluorescence emitted by the fluorescent material. The polymerase chain reaction amplifies the polynucleotide by repeatedly performing three steps of varying the temperature of denaturation, annealing, and elongation with the polynucleotide as dNTP, primer, and polymerase. Means. The real-time polymerase chain reaction may use an intercalating method, a TaqMan probe method, or a cycling probe method, depending on the fluorescent material used, but is not limited thereto.

The method comprises the steps of (a) performing a polynucleotide polymerase chain reaction on a plurality of samples comprising two or more identical samples, wherein the polymerase chain reaction comprises a plurality of amplification cycles, Performing in the presence of a detectable probe capable of providing a signal accordingly.

The plurality of samples refers to a plurality of samples including polynucleotides to be amplified by real time polymerase chain reaction. The plurality of samples may include polynucleotides having the same or different concentrations of each other, and may include polynucleotides having the same or different sequences from each other. In addition, the plurality of samples include the cytoplasm of cells taken from the same tissue, but may be different in the type of primer added according to the gene to determine the expression amount. For example, the plurality of samples may be taken from different kinds or cells of the same kind, and primers added may be different depending on the gene to be expressed.

In step (a), the plurality of samples include two or more identical samples. The same sample refers to samples having the same contents for performing a real-time polymerase chain reaction. That is, it means samples that contain the same polynucleotide sequence, type, concentration, and all the materials such as primers, dNTPs, fluorescent indicators, and the like necessary to perform real-time polymerase chain reaction. The inclusion of the same content in the same manner means that it is presumed to be substantially the same in kind and amount, as well as equivalent to those skilled in the art. For example, the same amount of cytoplasm taken from the same kind of cells cultured under the same conditions is considered to contain the same amount of polynucleotides although the amount of polynucleotides may vary substantially substantially from cell to cell.

The detectable probe is an intercalating dye such as SYBR Green I, Ethidium bromide, YO-PRO-1 BOXTO, labeled with donor fluorephore (FITC) at the 3 'end and acceptor fluorophore at the 5' end. (acceptor fluorophore) labeled fluorogenic hybridization oligoprobe, TaqMan prob, Hairpin oligoprobe, self-fluorescing amplicon (sunrise primer & scorpion) primers)), but not limited to these.

The method includes (b) obtaining a plurality of amplification profile curves for signal strength values provided by the probe according to the number of amplification cycles for each sample.

RT PCR increases the number of amplification cycles and increases the amount of polynucleotides and the signal strength value provided by the probe. The amplification profile curve refers to a relationship function that detects such signal strength values and indicates signal strength values provided by the probe according to the number of amplification cycles. Preferably, the amplification profile curve is obtained by dividing the detected signal strength by the signal strength from the passive reference dye, where the increase in the response initial signal strength value does not appear at the normalized value of Rn or Rn (baseline region). ΔRn after subtracting the Rn value of) is represented by the y-axis and the number of amplification cycles on the x-axis.

The method includes (c) selecting one threshold from the signal strength values.

The threshold may be a randomly selected one of the signal strength values applied in the amplification profile curve. Preferably, the threshold is a signal strength value between a baseline region and a stationary region in the amplification profile curve, more preferably a signal strength value at a point from the baseline region to an exponential region in the amplification profile curve. Can be. The baseline region is a region in which the initial signal strength value of the real-time PCR reaction does not continuously increase in the amplification profile curve, and the exponential region is a region in which the signal strength value increases in the amplification profile curve, the congestion state. An area means an area in which the signal intensity value that appears after the exponential area does not increase in the amplification profile curve. The baseline region appears because the amount of polynucleotide has not yet reached the polynucleotide amount with a detectable signal intensity value.

The method includes (d) calculating a threshold cycle value corresponding to the selected threshold value from the plurality of amplification profile curves.

The term "thresold cycle value" means the value of the number of amplification cycles when the signal strength value in the amplification profile curve is a threshold. The higher the initial polynucleotide amount, the more exponential regions begin at fewer amplification cycles. Therefore, the higher the initial polynucleotide amount, the smaller the threshold cycle value.

The method includes (e) calculating a variance value of threshold cycle values of the same samples.

Step (e) is a step of calculating the variance of the critical cycle values obtained from the amplification profile curves of the same samples calculated through steps (c) and (d). When the critical cycle values are {x ₁ , x ₂ , ..., x _n } and the average of these critical cycle values is m, the variance σ ² is as follows, which is well known to those skilled in the art.

Performing real-time polymerase chain reaction under the same conditions with a sample containing the same sample should theoretically produce the same amplification curve, but in reality it is difficult to produce a perfectly matching amplification curve. Therefore, the critical cycle values obtained from the same samples in step (d) may not all be the same, and thus the variance may not be zero.

The method includes (f) repeating steps (c), (d), and (e) a plurality of times by varying the threshold to select.

Repeating steps (c), (d), and (e) by selecting different thresholds each time will select several thresholds and yield threshold cycle values for each of the selected thresholds. The plurality of times is an integer number of times greater than or equal to zero determined by the user, and the larger the number of times, the greater the possibility of obtaining an optimal threshold value. Preferably, the threshold value may be differently selected a plurality of times in any region including the signal intensity value of the portion of the baseline region of the amplification profile curve that passes from the exponential region to the exponential region, or the region may be equally divided multiple times. You can select it as a threshold.

The method includes the step of (g) selecting the threshold value of the step (c) of calculating the smallest variance value among the variance values calculated in the step (e) as the final threshold value.

The variances calculated in step (e) are the variances between the threshold cycle values obtained from the same samples, and the critical cycle values obtained from the same samples should be identical in theory, so the smaller these values are the threshold cycle values representing the actual amount of polynucleotides. can do. Therefore, the threshold having the smallest variance of the threshold cycle values of the same samples is selected as the final threshold.

A second aspect of the present invention is a method of selecting a threshold value from an amplification profile curve obtained by performing a real-time polymerase chain reaction.

(a) performing a polynucleotide polymerase chain reaction on a plurality of samples including two or more identical samples, wherein the polymerase chain reaction includes a plurality of amplification cycles and provides a signal according to the amount of polynucleotides. Performing in the presence of a detectable probe that can provide;

(b) acquiring a plurality of amplification profile curves for signal strength values provided by the probe according to the number of amplification cycles for each sample;

(c) selecting one threshold from the signal strength values;

(d) calculating a threshold cycle value corresponding to the selected threshold value from the plurality of amplification profile curves;

(e) calculating a variance value of threshold cycle values of the same samples;

(f) calculating a representative value from threshold cycle values of the same samples;

(g) calculating the variance values of the threshold cycle values of the remaining samples except for the same samples and the representative value calculated in step (f);

(h) calculating a value obtained by dividing the variance value calculated in step (e) by the variance value calculated in step (g);

(i) repeating steps (c), (d), (e), (f), (g), and (h) a plurality of times with different threshold values selected; And

(j) selecting the threshold value of the step (c) of calculating the smallest value among the values calculated in the step (h) as a final threshold value.

The method of the present invention provided by the second aspect is the same from step (a) to step (e) as the method provided by the first aspect of the present invention.

The method includes (f) calculating a representative value from threshold cycle values of the same samples.

Step (f) is a step of calculating a value representative of these of the critical cycle values obtained from the same samples, the method of calculating can be arbitrarily determined by the user. For example, it may be, but is not limited to, an average value, a median value, or any of these values of critical cycle values obtained from the same sample. The reason for calculating the representative value in the step (f) is to obtain the variance between the threshold cycle values of the different samples in the following step (g), where one threshold cycle value representing the same samples is distributed for the same samples. To be included in the calculation.

The method includes (g) calculating the variance values of the threshold cycle values of the remaining samples except for the same samples and the representative value calculated in step (f).

Step (g) is a step of calculating a variance value between threshold cycle values of different samples. If there are two or more identical samples, the representative value calculated in step (f) is the threshold cycle value for those same samples, and in the case of a sample in which two or more identical samples do not exist, in step (d) The variance between all the threshold cycle values is calculated by using the calculated threshold cycle value as each threshold cycle value as it is.

The method comprises the steps of: (h) calculating a value obtained by dividing the variance value calculated in step (e) by the variance value calculated in step (g), (i) varying a threshold value to be selected, and (c) repeating steps (d), (e), (f), (g), and (h) a plurality of times; And (j) selecting the threshold value of the step (c) of calculating the smallest value among the values calculated in the step (h) as the final threshold value.

The method of selecting the threshold value a plurality of times in step (i) is the same as described above for step (f) of the method provided by the first aspect of the present invention. In step (h), the variance calculated in step (e) (hereinafter referred to as intraclass variance) is the variance between threshold cycle values of the same sample and the variance calculated in step (g) (hereinafter referred to as interclass variance). Is the variance between the threshold cycle values of different samples. The intraclass variance is the variance between the threshold cycle values obtained from the same samples, and since the critical cycle values from the same sample should be the same in theory, the smaller this value, the better the threshold cycle values representing the actual polynucleotide amount. Interclass variance is the variance between the threshold cycle values of different samples including samples of different composition. Different samples may inadvertently contain similar concentrations of polynucleotides, but generally the amount of polynucleotides will be different. Therefore, the greater the interclass variance, the better the separation of different polynucleotide amounts and the more accurate polynucleotide quantification. In conclusion, the smaller the intraclass variance value, the closer the critical cycle values for the same polynucleotide amount are at the point of the threshold where the interclass variance value is higher, and the critical cycle values for the different polynucleotide amounts are far apart. Threshold cycle values that well reflect the amount of polynucleotides can be obtained and more accurate quantification of polynucleotides can be achieved when these thresholds are used for quantification. In order to satisfy this condition, in step (h), the value obtained by dividing the intraclass variance value by the interclass variance value is calculated, and the threshold value that calculates the smallest value among the values calculated in step (j) to (h) is final. The threshold will be selected. The present invention has similar characteristics to linear discriminant analysis (LDA), which is a method of finding a vector that maximizes the degree of separation of data in terms of finding a specific value that maximizes the variance between classes and minimizes the variance in the class.

Among the methods of selecting the existing threshold value, the method of using the differential coefficient calculates the threshold by differentiating for each amplification profile curve and selects the representative value as the threshold value. It was hard to see the features integrated. Since the method of the present invention considers all amplification profile curve data integrally, a result value reflecting the results of the entire experiment can be obtained. In addition, the overall experimental results are integrated to eliminate more noise that is inconsistent with the overall data flow.

According to one embodiment of the present invention, when two or more values are calculated in step (e), the dispersion value calculated in step (e) in step (g) or step (h) is step (e). It provides a method that is representative of the variance values calculated in.

In general, when the experiment is performed, the same experiment is repeated several times to increase the reliability and accuracy of the experiment. Even when real-time polymerase chain reaction is performed, the experiment may be performed by making two or more identical samples for each sample. That is, not only one set of identical samples but also several sets of identical samples (in which case, the samples between each set have different configurations of contents included in each other) may be used in the experiment. In this case, since there are a plurality of variances for the same sample in step (e), a plurality of variance values may be added to "variance values calculated in step (e)" corresponding to intraclass varience in step (g) or step (h). Substitute the representative value calculated from Representative values can be determined by the user and various methods well known in the art can be used. For example, the representative value can be the mean, median, or any of the variables.

One embodiment of the present invention provides a method in which the representative value calculated in step (f), (g) or (h) is an average value.

In one embodiment of the present invention, step (e) calculates a variance value of the threshold cycle values of the same samples, except for a threshold cycle value that differs from the average value of the threshold cycle values of the same samples by more than a predetermined value. It provides a method that is.

The critical cycle values from the same sample should be identical in theory, and the smaller the error between them, the more reliable the experiment. If any of the critical cycle values obtained from the same sample differ greatly from the other critical cycle values, this is a value that accurately reflects the amount of polynucleotides, rather than the experimenter's immaturity, contamination of the sample, or error in measuring equipment. Most likely noise. Therefore, calculating these variances, excluding these values, will help you choose a more accurate threshold. In this method, the variance is calculated by excluding the critical cycle values that differ by more than a predetermined value from the average value of the critical cycle values. The predetermined value may be arbitrarily set by the user as an error value that may be regarded as noise when the user thinks.

When using the present invention, even in the quantification of polynucleotides that do not use a standard curve, more accurate threshold values can be selected for accurate polynucleotide quantification.

Figure 1 shows a general amplification profile curve that can be obtained by performing a real-time polymerase chain reaction.
FIG. 2 shows amplification profile curves, thresholds, and thus threshold cycle values (CT) when real-time polymerase chain reactions were performed with different initial polynucleotide amounts.
Figure 3 shows the amplification profile curve obtained by performing a real-time polymerase chain reaction according to Example 1.
4 shows intraclass variance values, interclass variance values, and their ratios (intraclass variance values / interclass variance values) when ΔRn values of 0 to 5 are selected as threshold values in the amplification profile curve of S18.
Figure 5 shows the results obtained by subtracting the variance calculated using the Applied Biosystems 7500 Real-Time PCR machine from the variance calculated using the method of the present invention. A total of 24 variances are generated by calculating the variance between the thresholdcycle values of the same four samples calculated from the last threshold.

Example 1 Selection of Thresholds

Real-time polymerase chain reaction was performed to determine the relative expression levels of 18S, OGDH, EGR3, OSGEP, MAOB, and SERPING1 genes. The expression level of the 18S gene was used as a control.

In order to determine the expression level of OGDH gene, a pair of primers were added to extract the cytoplasm of cells in the rat brain, kidney, liver, and heart, and to amplify the mRNA of OGDH gene. In total, 16 samples of OGDH gene expression were made.

Samples were made in the same manner for the 18S, EGR3, OSGEP, MAOB, and SERPING1 genes, and real-time polymerase chain reaction was performed using Applied Biosystems' 7500 Real-Time PCR machine using a total of 96 samples. The number of amplification cycles was 40 and the fluorescent probe used was a TaqMan probe. The amplification profile curve obtained by performing the real-time polymerase chain reaction under the above conditions is shown in FIG. 3 (the value of the y-axis (signal intensity value) is represented by ΔRn).

From the data of 16 amplification profile curves regarding the amount of OGDH gene expression, a threshold cycle value was calculated using each ΔRn value having a value of 0 to 5 as a threshold. The variances between the critical cycle values of the same samples were obtained (one variance was calculated for each tissue, resulting in a total of four variances) and their average was taken as the intraclass variance value. The average value of the critical cycle values of the same samples for each tissue was calculated (the average value was calculated for each tissue, and a total of four average values were calculated). The average value was used as the representative value of the tissue. The intraclass variance value was divided by the interclass variance value and the threshold when the value was the smallest was selected as the final threshold value. The intraclass variance value, interclass variance value, and their ratio (intraclass variance value / interclass variance value) when ΔRn of 0 to 5 are selected as thresholds are shown in FIG. 4. Final thresholds were selected for each gene in the same manner as above, and the final thresholds of the 18S, OGDH, EGR3, OSGEP, MAOB, and SERPING1 genes were 0.7000, 0.4000, 0.2000, 0.2000, 0.1000, and 0.2000.

The same intersample variance (24 sets of 4) at the final thresholds calculated using the method of the present invention was smaller than the variance calculated using the 7500 Real-Time PCR machine from Applied Biosystems (FIG. 5). Since the same four samples should theoretically have the same threshold cycle value, it can be seen that the method of the present invention, which selects an optimal threshold with a smaller variance between identical samples in the amplification profile curve, allows for more accurate quantification.

[Matlab code used to perform Example 1]

function [Threshold CtList] = comparative_Ct (deltaRn, sampleIndex, numSamples, eachIndex)

numCycles = 40;

mx = max (max (deltaRn (:, sampleIndex)));

ThresholdList = 0.1: 0.1: mx;

thresholdLength = length (ThresholdList);

CtMatrix = zeros (thresholdLength, length (sampleIndex));

for wellIndex = 1: length (sampleIndex)

    CtMatrix (:, wellIndex) = compCtMatrix (deltaRn, ThresholdList, sampleIndex (wellIndex), numCycles);

end

averageSample = zeros (thresholdLength, numSamples);

var_List = zeros (thresholdLength, numSamples);

for sampleId = 1: numSamples

    index = eachIndex (sampleId, :);

    averageSample (:, sampleId) = mean (CtMatrix (:, index), 2);

    var_List (:, sampleId) = var (CtMatrix (:, index), [], 2);

end

[X I] = min (sum (var_List, 2) ./ var (averageSample, [], 2));

Threshold = ThresholdList (I);

CtList = CtMatrix (I, :);

Claims (6)

A method of selecting a threshold value from an amplification profile curve obtained by performing a real-time polymerase chain reaction, wherein the method
(a) performing a polynucleotide polymerase chain reaction on a plurality of samples including two or more identical samples, wherein the polymerase chain reaction includes a plurality of amplification cycles and provides a signal according to the amount of polynucleotide Performing in the presence of a detectable probe that can provide;
(b) obtaining an amplification profile curve for the signal strength value provided by the probe according to the number of amplification cycles for each sample;
(c) selecting one threshold from the signal strength values;
(d) calculating a threshold cycle value corresponding to the selected threshold value from the amplification profile curves;
(e) calculating a variance value of threshold cycle values of the same samples;
(f) repeating steps (c), (d), and (e) a plurality of times by varying the threshold value to be selected; And
and (g) selecting the threshold value of step (c) of calculating the smallest variance value among the variance values calculated in step (e) as the final threshold value. A method of selecting a threshold value from an amplification profile curve obtained by performing a real-time polymerase chain reaction, wherein the method
(a) performing a polynucleotide polymerase chain reaction on a plurality of samples including two or more identical samples, wherein the polymerase chain reaction includes a plurality of amplification cycles and provides a signal according to the amount of polynucleotides. Performing in the presence of a detectable probe that can provide;
(b) acquiring a plurality of amplification profile curves for signal strength values provided by the probe according to the number of amplification cycles for each sample;
(c) selecting one threshold from the signal strength values;
(d) calculating a threshold cycle value corresponding to the selected threshold value from the plurality of amplification profile curves;
(e) calculating a variance value of threshold cycle values of the same samples;
(f) calculating a representative value from threshold cycle values of the same samples;
(g) calculating the variance values of the threshold cycle values of the remaining samples except for the same samples and the representative value calculated in step (f);
(h) calculating a value obtained by dividing the variance value calculated in step (e) by the variance value calculated in step (g);
(i) repeating steps (c), (d), (e), (f), (g), and (h) a plurality of times with different threshold values selected; And
(j) selecting the threshold value of the step (c) of calculating the smallest value among the values calculated in the step (h) as a final threshold value. The method of claim 1, wherein when two or more values are calculated in the step (e), the variance value calculated in the step (e) in the step (g) is representative of the variance values calculated in the step (e). The value. The method of claim 2, wherein when two or more values are calculated in step (e), the variance value calculated in step (e) in step (h) is representative of the variance values calculated in step (e). The value. The method of claim 2, wherein the representative value calculated in step (f) is an average value. The method of claim 1 or 2, wherein step (e) calculates a variance value of the threshold cycle values of the same samples, except for a threshold cycle value that differs from the average value of the threshold cycle values of the same samples by more than a predetermined value. Step.

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