At least six replicates per condition for all experiments. At least 12 replicates per condition for experiments where identifying the majority of all DE genes is important. For experiments with <12 replicates per condition; use edgeR (exact) or DESeq2.
Table of Contents
How many biological replicates are needed in qPCR?
As mentioned by Udvardi et al. (2008), an experiment ideally should encompass at least three independent biological replicates of each treatment. For each biological replicate, it is common to run at least two technical replicates of each PCR reaction.
Can you combine biological and technical replicates?
Combine each technical replicates for each biological replicate, then combine each biological replicate into a group (i.e. treated group or baseline group). Remember technical replicates are there for measuring variability resultant from pipetting, whilst biological replicates for biological variation in expression.
Why are there 3 biological replications?
[3] Biological replicates are important because they address how widely your experimental results can be generalized. They indicate if an experimental effect is sustainable under a different set of biological variables.
How many biological replicates are needed for RNA-seq?
For future RNA-seq experiments, these results suggest that at least six biological replicates should be used, rising to at least 12 when it is important to identify SDE genes for all fold changes.
How many technical replicates should I have?
As for technical replicates, usually you will need 3 for each biological sample (also for positive and negative controls), and in a pinch it may be reduced to 2. You basically only need them to make sure that your reaction is reproducible.
How can I improve my qPCR replicates?
This increase could be accomplished by increasing the sample volume delivered or using more concentrated samples. Another approach is to increase the number of replicates, which reduces the impact of low copy variability on the mean.
How close should qPCR replicates be?
For a dataset that resulted from the qPCR measurement of 12 miRNA targets in 834 patients with a total of 20,016 reactions, the strict application of the rule that the Cq values of replicates should be within 0.5 cycles led to rejection of 7752 (39%) measurements.
Why is an increased number of replications an advantage in an experiment?
Quality is often more important than quantity. Science relies heavily on replicate measurements. Additional replicates generally yield more accurate and reliable summary statistics in experimental work.
What is the difference between technical replicate and biological replicate?
Generally, biological replicates are defined as measurements of biologically distinct samples that show biological variation (21). In contrast, technical replicates are repeated measurements of the same sample that show independent measures of the noise associated with the equipment and the protocols.
How do you combine data from repeated experiments?
If you repeat the experiments on the same sample unit, you can combine the experiments as technical replication. If, on the other hand, each experiment repetition is performed on different sample units, then you can combine all experiments as one bigger experiment.
Should you average technical replicates?
Averaging technical replicates (as in the left panel) and running statistical analyses on average values means losing potentially important information. No facet should be dropped from analysis unless one is confident that it can have absolutely no effect on analyses.
How many replicates should a well designed experiment include?
Please note that replications should be at least 2. The more you do replications, the more precise results you get.
Why do you run your samples in triplicate?
Triplicates in scientific experiments are important to validate empirical data or the observed results. In general, a research plan entails three replicates so that the results obtained from them can be verified. Thus, the relative differences of data from the three replicates can be measured and compared.
What is the difference between replicate and duplicate?
The word duplicate is derived from the Latin word duplicare, which means to double. Replicate means to reproduce something, to construct a copy of something, to make a facsimile. The word replicate may be interchangeable with the word duplicate except in a few instances.
Why is it important to have biological replicates in an RNA-seq experiment?
Biological variability is usually the largest effect limiting the power of RNA-seq analysis. The most improvement in an experiment will usually be achieved by increasing the biological replication to improve estimation of the biological variation.
What is a good number of reads for RNA-seq?
The number of reads required depends upon the genome size, the number of known genes, and transcripts. Generally, we recommend 5-10 million reads per sample for small genomes (e.g. bacteria) and 20-30 million reads per sample for large genomes (e.g. human, mouse).
What are technical and biological replicates?
Broadly speaking, biological replicates are biologically distinct samples (e.g. the same type of organism treated or grown in the same conditions), which show biological variation; technical replicates are repeated measurements of a sample, which show variation of the measuring equipment and protocols.
What counts as a biological replicate?
Biological replicates are parallel measurements of biologically distinct samples that capture random biological variation, which may itself be a subject of study or a source of noise.
Are biological replicates independent experiments?
As biological experiments can be complicated, replicate measurements are often taken to monitor the performance of the experiment, but such replicates are not independent tests of the hypothesis, and so they cannot provide evidence of the reproducibility of the main results.
What are different types of replicates?
There are two primary types of replicates: technical and biological.
Can I freeze qPCR plate?
Not ideal to make the reaction and do it few days later, unless you are running of the reagent and you really need that result. (Should you be in that situation again, consider freezing the plate (at least -4*C) rather than fridge storage as it may be more stable this way.
Should I dilute my cDNA?
It is necessary to dilute the cDNA sample, since for most genes the cDNA is too concentrated for qPCR.
How many times can you freeze thaw cDNA?
If your controls go wrong, just throw the aliquot away and use another. Dear Sungeun, if the cDNA (concentrated or diluted both) is kept at -20 to -35 C for more than a month, or freeze thawing a same aliquot more than 3 times so this same sample wont give the same result in qPCR.
How much DNA do I need for qPCR?
Use up to 100 ng of genomic DNA or 10โ107 copies of plasmid DNA in a 10-ฮผl volume.