Question: Replication in PCR-array experiments
0
gravatar for aaozmen
5.2 years ago by
aaozmen0
Turkey
aaozmen0 wrote:

Is it enough to use only three biological replicates  for correct statistics PCR-array analysis or three biological reps and two  technical reps or one biological rep. and three tech rep?

 

Our study  divided into a total of 3 main groups and 15 subgroups.  Each main groups contains 5 subgroups. these subgroups associated with time (1h, 2h,3h, 4h and 5h). Like this:

Group A: Control                                                     Group C: Treatment Group

subgroup I, (1h)...........3 rats                                          subgroup I, (1h)...........3 rats                    

subgroup 2  (2h)...........3 rats                                        subgroup 2  (2h)...........3 rats

.....                                                                            .....

subgroup 5 (5h)..........3 rats                                         subgroup 5 (5h)..........3 rats

 

Group B: Negative Control

subgroup I, (1h)...........3 rats

subgroup 2  (2h)...........3 rats

.......

subgroup 5 (5h)..........3 rats     

My questions:

3 rats in each group are biological reps. (I think). Are biological reps enough? or are techinical reps necessary? How many is enough for technical replicates?

and

At the end of the project, I will have results of each animals' 88 gen expression. Located timing in each sub-group is important, too.  What program should I use to compare differences in gene expression? and how can create a heat map?

 

ADD COMMENTlink modified 5.2 years ago by Carlo Yague5.5k • written 5.2 years ago by aaozmen0
1
gravatar for Carlo Yague
5.2 years ago by
Carlo Yague5.5k
Canada
Carlo Yague5.5k wrote:

Biological replicates can be enough if the only question you are interested in is identifying DEG, i.e, comparing the variability between conditions with the variability between biological replicates (that includes both technical and biological variability).

But technical replicates allows to differentiate technical from biological variability and to know if something went wrong during the experiment. So technical replicates are not as essential as biological ones, but since it costs you less (in term of work, money and stuff), you might as well includes them. They can also increase the power of your tests to identify DEG. How many technical reps is up to you, you need to find the balance between cost and benefit. I wouldn't have more than three though.

To find DEG, you can use "R" or perhaps Excel.

For heatmap, you can use "R" or cluster with cluster and treeview.

ADD COMMENTlink modified 5.2 years ago • written 5.2 years ago by Carlo Yague5.5k
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