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					            BIO 151 Laboratory, Digit ratio and testosterone levels
Before coming to lab, in the space below, write a step-by-step bulleted protocol for reproducibly and
reliably measuring 2D:4D ratio, based on the method used in, Honekopp J, T Manning J, Muller C.
Digit ratio (2D:4D) and physical fitness in males and females: Evidence for effects of prenatal
androgens on sexually selected traits. Horm Behav. 2006 Apr;49(4):545-9, which can be found at:
                                                                                                   Digit ratio and testosterone levels


         In 1983 Dr Glenn Wilson published a paper suggesting a correlation between assertiveness and
         2D:4D digit length in women1. The 2D digit is the index finger and the 4D digit is the ring
         finger. In 1998 John Manning published a paper showing a correlation between 2D:4D ratio
         and in-utero sex hormone exposure2. Since then Manning and others have published
         controversial work connecting 2D:4D ratio to athleticism and homosexuality34.

         The 2D:4D ratio is a sexually dimorphic trait, the average phenotypes of the two genders differ.
         The 2D:4D ratio in women tends to be 1 or slightly greater than 1 and in men it tends to be
         slightly less than 1. It should be noted that the magnitude of this effect is very small when
         compared to other known sexually dimorphic traits in humans like height.

         It is now generally accepted that digit ratio is roughly inversely correlated with embryonic
         testosterone exposure and directly correlated with in utero estrogen exposure. Because of the
         potentially profound effects sex hormones have on the developing fetus, and the growing
         realization that what happens in utero can have affects into late adulthood5, attempts have been
         made to link 2D:4D ratio to a number of physiological and behavioral traits (Table 1).

           Table 1 Some Characteristics That May Be Associated with Digit Ratio (from Manning, 2002b)
                                 Low 2D:4D ratio                         High 2D:4D ratio
                         More fertile                        Higher risk of early heart disease
                         Higher lifetime reproductive
            Males          success
                         More aggressive and assertive
                         Greater proclivity toward
                         Higher musical and sports aptitude
                         Greater proclivity toward            More fertile
            Females        homosexuality/bisexuality           Higher lifetime reproductive
                         More aggressive and assertive           success
                                                               Higher risk of breast cancer

         In today’s lab I would like us to examine some of the methodologies involved in studies like
         this. The class will design and carry out our own 2D:4D study. The study will seek a
         correlation between 2D:4D ratio and testosterone levels in college age men and women. A
         procedure for determining testosterone levels from spit follows. But each group will decide on
         a protocol for measuring 2D:4D ratio. Each group will then measure the 2D:4D ratio for every
         member of the class. We will also collect testosterone level data for the class and see if there is
         a correlation.

  Wilson, G.D. (1983). Finger-length as an index of assertiveness in women. Personality and Individual Differences 4, 111-112.
  Manning JT, Scutt D, Wilson J, Lewis-Jones DI. The ratio of 2nd to 4th digit length: a predictor of sperm numbers and concentrations
of testosterone, luteinizing hormone and oestrogen. Hum Reprod. 1998 Nov;13(11):3000-4.
  Manning JT. The ratio of 2nd to 4th digit length and performance in skiing. J Sports Med Phys Fitness. 2002 Dec;42(4):446-50.
  Robinson SJ, and Manning JT. The ratio of 2nd to 4th digit length and male homosexuality. Evol Hum Behav. 2000 Sep 1;21(5):333-
  Godfrey KM, Barker DJ. Fetal nutrition and adult disease. Am J Clin Nutr. 2000 May;71(5 Suppl):1344S-52S.

                                                                        Digit ratio and testosterone levels


Measuring 2D:4D ratio:
     Use the space below to write a protocol to sample the class for 2D:4D ratio. Spend some time
     discussing this with your group and thinking about the protocol you diagrammed for the pre-lab
     before writing your protocol.

                                                                          Digit ratio and testosterone levels

Testosterone Determination:
       We will be performing a Testosterone (Direct Saliva) ELISA assay. The kit we will be using to
       perform this assay is from Alpco Immunoassays. The complete manual for this kit can be found

Specimen collection and storage:
      Approximately 1 ml of saliva is required per duplicate determination. Collect 2-3 ml of saliva
      into a clean glass tube without force or inducement and before eating, drinking or brushing the
      teeth. Simply rinse the mouth with water before collection. Store samples at 4oC for up to 24
      hours or at -10oC or lower if the analyses are to be done at a later date. Consider all human
      specimens as possible biohazardous materials and take appropriate precautions when handling.

Specimen pretreatment:
   1. Place each sample into the benchtop centrifuge. Centrifuge the sample at 2000 rpm for 10
   2. Transfer 500 µl of the supernatant from the top to a new glass tube (13 mm X 100mm).
   3. Place the tubes containing the supernatant in a 65°C waterbath for 1 hour.
   4. Allow the heated samples to reach room temperature before assaying.

   Specimen samples should be assayed in duplicate. Once the procedure has been started, all steps
   should be completed without interruption.

   1. Remove the required number of microwell strips. Reseal the bag and return any unused strips to
       the refrigerator.
   2. Pipette 100 µl of each specimen sample into correspondingly labeled wells in duplicate.
   3. Pipette 100 µl of the testosterone-biotin conjugate working solution into each well.
   4. Incubate on a plate shaker (approximately 200 rpm) for 1 hour at room temperature.
   5. Wash the wells 5 times with 300 µl of diluted wash buffer per well and tap the plate firmly
       against absorbent paper to ensure that it is dry.
   6. Pipette 150 µl of the streptavidin-HRP conjugate working solution into each well.
   7. Incubate on a plate shaker (approximately 200 rpm) for 30 minutes at room temperature.
   8. Wash the wells again in the same manner as step 6.
   9. Pipette 150 µl of TMB substrate into each well at timed intervals.
   10. Incubate on a plate shaker for 10-15 minutes at room temperature (or until calibrator A attains
       dark blue color for desired OD).
   11. Pipette 50 µl of stopping solution into each well at the same timed intervals as in step 10.
   12. Read the plate on a microwell plate reader at 450 nm within 20 minutes after addition of the
       stopping solution.

                                                                                        Digit ratio and testosterone levels

       Complete the following table pooling data from the entire class.
        Subject #      2D:4D ratio       concentration

       Enter the data from the table above into an Excel spreadsheet and use Excel to draw an XY (Scatter) plot of the
       data. Add a trendline to the plot and perform a regression analysis on the data. We’ll do all this together.

Lab Report: (No handwritten reports please.)

       Your write-up for this lab should include:
          • An introduction that describes the rationale for asking this particular question. Why
             does it make sense and why is it interesting?
          • A results section that includes:
                 o The Excel Spreadsheet with the raw data and regression analysis on it.
                 o The graph you generated in Excel with the trendline on it.
                 o Any other graphs and statistical analyses we did wit the data.
          • A discussion that evaluates your data. Is there a correlation? Is it strong? What
             confounding factors might there be?